NUCLEIC ACID AGENTS MODULATING PD-1 ISOFORMS

FIELD OF THE INVENTION

The invention relates to nucleic acid agents modulating the expression of PD-1 isoforms, and methods of using same in immunotherapy and immunomodulation.

BACKGROUND OF THE INVENTION

Programmed cell death 1 receptor (PD-1) is an immune checkpoint receptor expressed on the surface of activated T, natural killer (NK) and B lymphocytes, macrophages, dendritic cells (DCs) and monocytes. It is categorized as a type I transmembrane protein, structurally belonging to the CD28/CTLA-4 subfamily of the Ig superfamily, and is encoded by the PDCD1 gene. PD-1 and its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC) belong to the immune checkpoint pathway, which induces immune suppression. The binding of PD-1 to its ligand (e.g. PD-L1) downregulates immune reactivity and promotes self-tolerance by suppressing the activity of effector T cells. PD- L1 is also expressed by many tumor cells, thereby facilitating their evasion from immune surveillance and down-regulating anti-tumor immunity.

Alternative splicing is a post- transcriptional process by which precursor mRNAs (pre- mRNAs) are spliced differentially, leading to distinct mRNA and protein isoforms, thus increasing the diversity of the human transcriptome and proteome. Dysregulation of splicing and alternative splicing underlies many genetic and acquired diseases. In addition, alternative splicing of immune receptors can potentially generate isoforms with a changed modulatory effect compared to the full- length receptor.

Alternative splicing is naturally regulated by cis-acting elements within pre-mRNAs and trans-acting factors. The essential cis-acting elements are the 5' splice site, the 3' splice site, as well as the branchpoint sequence, which conform to partially conserved motifs that are recognized by cognate trans-acting factors. However additional cis-acting elements that regulate alternative splicing are known, including exonic or intronic splicing enhancers and silencers (ESEs, ISEs, ESSs, ISSs), which respectively activate or repress use of particular splice sites or inclusion of exons.

Splicing modulation may also be achieved using exogenously-administered small molecules and nucleic acid agents using e.g. antisense or genetic engineering-based technologies. Antisense oligonucleotides (ASOs) are synthetic molecules comprised of nucleotides or nucleotide analogues that bind to a complementary sequence through Watson-Crick base-pairing. Although all ASO approaches make use of short nucleic acids that specifically base-pair to a targeted sequence, the outcome of such base-pairing depends on the chemistry of the oligonucleotide and the binding location. Splice-switching antisense oligonucleotides (SSOs) are ASOs that are typically 15-30 nucleotides long and designed to base-pair and create a steric block to the binding of splicing factors to the pre-mRNA. In this way, SSO base-pairing to a target RNA alters the recognition of splice sites by the spliceosome, which leads to an alteration of normal splicing of the targeted transcript (Havens et al., Nucleic Acids Research, 2016, Vol. 44, No. 14 6549-6563).

Typically, nucleotides of an SSO are chemically modified so that the RNA-cleaving enzyme RNase H is not recruited to degrade the pre-mRNA-SSO complex. Thus, SSOs modify splicing without necessarily altering the abundance of the mRNA transcript. The RNAse H- resistant features of SSOs are considered important, as the goal of SSOs is to alter splicing and not to cause the degradation of the bound pre-mRNA, unlike other antisense or silencing-based approaches. Various SSO strategies have been demonstrated to be effective in modulating splicing in animal models of human disease, and some have entered clinical trials, for example in the treatment of pediatric genetic disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy (Havens et al., ibid).

Ceccarello et al., (RNA BIOLOGY 2019, VOL. 16, NO. 12, 1794-1805) reported the design of SSOs as a tool to transiently modify T cell function. In particular, SSOs directed to three T cell-specific genes, namely interferon-y (IFN-y), perforin (PRF), and granzyme B (GZMB) were generated and examined. US 2020/0377883 relates to antisense oligonucleotides for modulating the function of a T cell, including antisense oligonucleotides that hybridize to IFN-y, granzyme, perforin 1, PD-1, PRDM1, PD-L1, CD40LG, NDFIP1, PDCD1 LG2, REL, BTLA, CD80, CD160, CD244, LAG3, TIGIT, AD0RA2A & TIM-3 RNAs. In particular, the publication relates to ASOs capable of inducing exon skipping of RNA. Sun et al. (RNA BIOLOGY 2019, VOL. 16, NO. 12, 1794-1805) examined the modulation of PDCD1 exon 3 splicing using a minigene system and a screen of PDCD1 -specific ASOs.

Gene editing (including genomic editing) is a type of genetic engineering in which nucleic acids are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell. In general, the technology achieves gene manipulation by artificially realizing double- stranded DNA breaks and using the repair mechanism of double- stranded DNA breaks. Available gene editing technologies include e.g. ZFN, TALEN and CRISPR/Cas9 technologies, of which CRISPR/Cas9 technologies are the most widely used.

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences found in the genome of prokaryotes, derived from DNA fragments of bacteriophages that had previously infected the prokaryote, used to detect and destroy DNA from similar bacteriophages during subsequent infections. CRISPR-associated protein 9 (Cas9) is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Gene editing systems generated based on CRISPR-Cas technology contain a guide RNA (gRNA, e.g. single guide RNA - sgRNA) and Cas protein. The gRNA is a short synthetic RNA composed of user-defined ~20 nucleotide sequence that targets the genomic DNA to be modified (target-binding sequence) and a scaffold sequence necessary for Cas-binding. The target-binding sequence pairs with the DNA target, directly upstream of a requisite 5'-NGG adjacent motif (PAM). Upon binding, Cas9 mediates a double strand break (DSB) ~3 bp upstream of the PAM.

Alternative splice variants of human PD-1 have been identified in peripheral blood mononuclear cells (PBMC; Nielsen et al., Cellular Immunology 235 (2005) 109-116), and more recently soluble forms of PD-1 and PD-L1 (sPD-1 and sPD-Ll) have been detected in the blood of patients with tumors. Increasing evidence suggests that the blood levels of sPD-l/PD-Ll might facilitate the prediction of clinicopathological characteristics, treatment response, and survival outcomes in patients with cancer. In addition, five splice variants of PD-1 mRNA transcripts have been cloned from human peripheral blood mononuclear cells: full-length PD-1 (flPD-1), PD-1 Deltaex2, PD-1 Deltaex3, PD-1 Deltaex2,3, and PD-1 Deltaex2,3,4. PD-1 Deltaex2 and PD-1 Deltaex3 are generated by alternative splicing where exon 2 and 3 (extracellular IgV-like domain and transmembrane domain, respectively) are spliced out. PD-1 Deltaex2,3 lacks exon 2 and 3. PD-1 Deltaex2,3,4 lacks exon 2, 3, and 4 (intracellular domain) and includes a premature stop codon in exon 5. PD-1 Deltaex2, PD-1 Deltaex2,3, and PD-1 Deltaex2,3,4 cannot bind to their ligands because of the lack of exon 2. The PD-1 Deltaex3 variant (lacking exon 3) encodes the sPD-1, which is up-regulated under certain pathological conditions, can retain PD-L1 binding domain and may compete with PD-1 for PD-L1 -binding, thus potentially resulting in a checkpoint inhibitor-like functionality.

Zhang et al. (Cytotherapy 22 (2020) 734-743) report on the generation of Chimeric antigen receptor (CAR) T cells, genetically modified to secrete constitutively soluble types of human PD- 1. The structure of CAR with sPD-1 was constructed by fusion of the second CAR and a truncated extracellular PD-1 (AA21-155) using a self-cleaving 2A peptide (T2A).

Checkpoint-blockade therapy seeks to reinvigorate T cell response in exhausted or otherwise hypofunctional tumor infiltrating lymphocyte (TIL) populations, by targeting inhibitory receptors known as immune checkpoint molecules, which are upregulated by these cells. Targeting immune checkpoints, including programmed death 1 (PD-l)/programmed death ligand 1 (PD-L1), has been demonstrated to be effective in treating advanced malignancies in some patients, and PD-1/PD-L1 inhibitors have been approved by the Food and Drug Administration (FDA) for treating melanoma, non-small cell lung cancer (NSCLC) and other malignancies. For example, Robert et al (Eur J Cancer. 2021 Feb; 144:182-191) and Reck et al (J Clin Oncol. 2021 Jul 20;39(21):2339-2349) examine the use of Pembrolizumab (Keytruda®), a humanized PD-l-blocking antibody, in advanced melanoma and metastatic non-small-cell lung cancer, respectively.

However, despite the impressive clinical success of checkpoint blockade therapy in a subset of selected patients, intrinsic or acquired tumor resistance remains a great challenge, leading to low response rate in large-scale use of immune checkpoint inhibitors in solid tumors.

European Publication No. 3498846 relates to artificially engineered immune regulatory factors and immune cells comprising same. The immune regulatory factor may be, for example, a genetically engineered or modified PD-1, CTLA-4, TNFAIP3(A20), DGKA, DGKZ, FAS, EGR2, PPP2R2D, TET2, PSGL-1, or KDM6A gene. In particular, the publication discloses the use of gene editing agents for suppressing or inactivating the immune regulatory gene.

US Publication No. 2019/0381157 relates to compositions (in particular, vaccine compositions) comprising at least one immune checkpoint antagonist and at least one antigen, and to methods of using same for modulating the immune system of a patient in need thereof. The immune checkpoint antagonist may be an antibody or soluble receptor molecule capable of binding an immune checkpoint associated protein, or a CRISPR-CAS9 encoding nucleic acid sequence capable of impairing or eliminating expression of an immune checkpoint associated protein selected from the group consisting of PD-L1, PD-L2, CD80, CD86, ICOS Ligand, B7-H3, B7-H4, 4-1BBL, HVEM, OX40L,CD70, CD40L, Galectin-9, Adenosine, GITRL, IDO, TDO, CEACAM1, VISTA, CD47, CD155, CD48, HHLA2, BTN2A1, BTN2A2, BTN3A1, BTNL3, BTNL9, PD-1, CTLA-4, CD28, ICOS, 4-1BB, BTLA, CD160, LIGHT, LAG3, 0X40, CD27, CD40, TIM-3, Adenosine A2a receptor, GITR, CEACAM1, SIPR-alpha, DNAM-1, TIGIT, CD96, 2B4, TMIGD2, and DC-SIGN. Additional approaches suggesting the use of gene editing to knock-out or eliminate the expression of target genes in their entirety (inter alia PD-1) in immune cells, including in particular CAR-T cells and other engineered cells, include, for example, WO 2015/161276, WO 2017/093969, WO 2022/094329, US 2019/0136230, US_10,729,725, US 2022/0162555, WO 2021/062227, WO 2022/086846, WO 2022/159753, WO 2022/067089, WO 2019/78225, WO 2020/16812, US 2022/0133790, WO 2022/011232, WO 2022/020785, US 2020/0224163, WO 2019/076489 and WO 2019/076486.

International Patent Application WO 2018/077189 discloses a high-affinity soluble PD-1 molecule that can recognize the PDL-1 molecules with high affinity. The high-affinity PD-1 molecule contains one or more mutations in the amino acid sequence. Specifically, the mutated amino acid residue positions in the high-affinity PD-1 molecule includes one or more of 91G, 3 IV, 33N, 35Y, 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 92A, 931, 95L, 97P, 98K, 99A, 100Q, 1011, and 103E.

International Patent Application WO 2012/062218 relates to soluble PD-1 proteins and nucleic acids, and therapeutic compositions comprising same, for enhancing immunity of a subject. The proteins, nucleic acids, and compositions may be formulated as a vaccine composition and include in particular soluble PD-1 variants comprising: mspdl-14del (deleting amino acids 26- 39 encoded by the first part of the second exon of the wild-type mouse PD-1 gene), mspdl-322mu (changing amino acid residue 108 of the wild-type mouse PD-1 protein from Met to Vai) and hspdl-14del (derived from a natural isoform of human PD-1, has a deletion of amino acids 26-39 encoded by the first part of the second exon of the wild-type human PD-1 gene).

There remains an unmet medical need for additional effective and safe therapeutic modalities for cancer.

SUMMARY OF THE INVENTION

The invention relates to nucleic acid agents modulating the expression of PD-1 isoforms, and methods of using same in immunotherapy and immunomodulation. Specifically, provided are nucleic acid molecules, including in particular splice-switching gene-editing agents, nucleic acid constructs encoding them, and methods of using same. The invention further relates to uses of the advantageous oligonucleotides, constructs and gene-editing systems in the preparation of cell compositions for immunotherapy.

The invention is based, in part, on the discovery of nucleic acid agents that are exceptionally effective in modifying the expression of PD-1 isoforms and improving anti-tumor immunity. Specifically, PD-1 -specific CRISPR/Cas9 editing systems were generated, and introduced to various immune cells using viral vectors in combination with a Cas9 endonuclease, or as ribonucleic protein (RNP) complexes. The systems, targeting sequences at the intron-exon junction of exon 3, were found to be highly effective in differential modulation of PD-1 isoforms. In particular, PD-1 isoform-specific gene editing systems generated were found to down-regulate the expression of full-length PD-1, while up-regulating the secretion of soluble PD-1 (sPD-1) in peripheral blood mononuclear cells (PBMC), T cell lines and tumor-infiltrating lymphocytes (TIL). Further, the manipulation resulted in remarkably enhanced T cell functionality, and enhanced activation-induced IL-2 secretion and tumor- specific cytotoxicity. This unique modulation pattern and associated functional manifestations was found to be distinct from those associated with previously tested approaches, such as conventional gene editing systems aimed at eliminating or downregulating the expression of PD-1 in a non-differential manner.

Thus, and without wishing to be bound by a specific theory or mechanism of action, the system was found to be remarkably effective in inducing splice-switching and promoting exon 3- skipping in immune cells, in a highly specific manner. Accordingly, the invention further relates in embodiments thereof to the identification of a novel target sequence within the PDCD1 gene, amenable for enhancing splice- switching and exon 3 -skipping using nucleic acid agents and systems of the invention. Advantageous gene editing systems of the invention may thus be readily used in long-term therapeutic regimens, while substantially reducing the risk of resistance, and while not obligatorily depending on the expression of the receptor on immune cells and its ligand on tumor cells for their activity.

In some embodiments, the invention relates to PD-l-specific expression-modulating nucleic acid molecules, the molecules comprising a sequence (of typically 15-30 contiguous nucleotides) that is specifically hybridizable (hybridizes, or is capable of hybridizing, in a selective manner) with a nucleic acid target within the PD-1 transcript or gene. As disclosed and exemplified herein, an advantageous nucleic acid target has the nucleic acid sequence as set forth in SEQ ID NO: 1, as follows:

TTTGTGCCCTTCCAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGC (SEQ ID NO: 1). According to a particularly advantageous embodiment, the nucleic acid molecule is specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2, as follows: TCCAGAGAGAAGGGCAGAAG (SEQ ID NO: 2). In one aspect there is provided an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene-editing system, comprising, or encoding: (i) a synthetic guide RNA (gRNA) molecule and (ii) an RNA-guided DNA endonuclease enzyme, wherein the gRNA molecule is a programmed cell death protein 1 (PD-l)-specific expressionmodulating nucleic acid molecule that downregulates the expression of the full-length PD-1 isoform and up-regulates the expression of a non-membrane bound PD-1 isoform, and wherein said molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2.

In one embodiment, said gRNA molecule has a targeting sequence (crRNA) that is specifically hybridizable with the nucleic acid target, wherein the targeting sequence is selected from the group consisting of: SEQ ID NOs: 3, 15 and 17, as follows: CUUCUGCCCUUCUCUCUGGA (SEQ ID NO: 3); UUCUGCCCUUCUCUCUGGAA (SEQ ID NO: 15); and GGCACUUCUGCCCUUCUCUC (SEQ ID NO: 17).

In a particular embodiment, said targeting sequence is as set forth in SEQ ID NO: 3. In another particular embodiment, said targeting sequence is as set forth in SEQ ID NO: 15. In another particular embodiment, said targeting sequence is as set forth in SEQ ID NO: 17. In yet another particular embodiment, said targeting sequence is as set forth in SEQ ID NO: 15 or 17. Each possibility represents a separate embodiment of the invention.

In another embodiment, said gRNA molecule is a single guide RNA (sgRNA) molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 16 and 18, as follows:

CUUCUGCCCUUCUCUCUGGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 4);

UUCUGCCCUUCUCUCUGGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGc (SEQ ID NO: 16); and

GGCACUUCUGCCCUUCUCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 18).

In another embodiment, said sgRNA molecule is as set forth in SEQ ID NO: 4. In another embodiment, said sgRNA molecule is as set forth in SEQ ID NO: 16. In another embodiment, said sgRNA molecule is as set forth in SEQ ID NO: 18. In another embodiment, said sgRNA molecule is selected from the group consisting of SEQ ID NO: 16 and 18. Each possibility represents a separate embodiment of the invention. In another embodiment, the gRNA and the RNA-guided DNA endonuclease enzyme are complexed to form a ribonuclear protein (RNP) complex. In another embodiment, the geneediting system comprises a first nucleic acid sequence encoding the gRNA, and a second nucleic acid sequence encoding the RNA-guided DNA endonuclease enzyme. In other embodiments the endonuclease is Cas9 or Cpfl, wherein each possibility represents a separate embodiment of the invention. In another embodiment, there is provided a vector comprising the engineered non- naturally occurring gene-editing system of the invention. In another embodiment, said vector is a viral vector.

In another aspect, there is provided a pharmaceutical composition comprising the system, and optionally a pharmaceutically acceptable carrier, excipient or diluent.

In one embodiment, the pharmaceutical composition is for use in inducing or enhancing splice switching in PD-1 expressing cells. In another embodiment, the pharmaceutical composition is for use in the treatment of cancer in a subject in need thereof. In another embodiment, the subject is afflicted with a treatment-resistant tumor. In another embodiment, the subject is afflicted by a tumor characterized by PD-L1 over-expression. In another embodiment, the subject is afflicted by a PD-L1 non-expressing tumor. In another embodiment, the use comprises: preparing a cell composition for immunotherapy by a method comprising the step of introducing said system into a leukocyte population, and administering the resulting cell composition to said subject.

In another aspect, there is provided a method of inducing or enhancing splice switching in PD-1 expressing cells, comprising introducing into the cells an engineered, non-naturally occurring CRISPR gene-editing system, comprising, or encoding: (i) a synthetic gRNA molecule and (ii) an RNA-guided DNA endonuclease enzyme, wherein said gRNA molecule comprises 15- 30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2.

In various embodiments, said gRNA molecule has a targeting sequence (that is specifically hybridizable with the nucleic acid target) selected from the group consisting of: SEQ ID NOs: 3, 15 and 17, or is a sgRNA molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 16 and 18, wherein each possibility represents a separate embodiment of the invention. In another embodiment, said cells are selected from the group consisting of peripheral blood mononuclear cells, T cells, B cells, Natural killer cells, antigen presenting cells, dendritic cells and macrophages, wherein each possibility represents a separate embodiment of the invention. In another embodiment, the system is introduced into the cells in vitro or ex vivo. In another embodiment, the system is introduced into the cells in vivo. Each possibility represents a separate embodiment of the invention. In another embodiment, the resulting cells are formulated in the form of a cell composition for immunotherapy.

In another aspect, there is provided a method of preparing a cell composition for immunotherapy, comprising the step of introducing into a leukocyte population, an engineered, non-naturally occurring CRISPR gene-editing system, in an amount and under conditions suitable for inducing or enhancing splice switching in the leukocyte population, wherein the system comprises, or encodes: (i) a synthetic gRNA molecule and (ii) an RNA-guided DNA endonuclease enzyme, and wherein said gRNA molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2.

In some embodiments, said gRNA molecule has a targeting sequence selected from the group consisting of: SEQ ID NOs: 3, 15 and 17, or is a sgRNA molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 16 and 18. In other embodiments, the leukocyte population is a T cell population comprising CD8 ⁺ T cells, and the cells are expanded so as to obtain a T cell composition adapted for adoptive transfer immunotherapy comprising an effective amount of the resulting T cell population. Each possibility represents a separate embodiment of the invention.

In another embodiment, the method further comprises administering the composition to a subject in need thereof. In another embodiment the subject is afflicted with a tumor. In another embodiment, the tumor is treatment-resistant. In another embodiment, said subject is afflicted by a tumor characterized by PD-L1 over-expression. In another embodiment, said subject is afflicted by a PD-L1 non-expressing tumor. In another embodiment, said tumor is selected from the group consisting of melanoma, renal cell carcinoma, lung cancer, breast cancer, head and neck cancer, bladder cancer, urothelial cancer, pancreatic cancer and cervical cancer. Each possibility represents a separate embodiment of the invention.

In another aspect there is provided a cell composition for immunotherapy, prepared by the method comprising the step of introducing into a leukocyte population, an engineered, non- naturally occurring CRISPR gene-editing system, in an amount and under conditions suitable for inducing or enhancing splice switching in the leukocyte population, wherein the system comprises, or encodes: (i) a synthetic gRNA molecule and (ii) an RNA-guided DNA endonuclease enzyme, and wherein said gRNA molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2. In another embodiment there is provided a cell composition for immunotherapy, prepared by the method as disclosed herein. In another embodiment, at least a portion of the cells are PD-1 -expressing cells characterized by a splice switching indel mutation, the mutation associated with non-homologous end-joining (NHEJ) repair of a double- stranded DNA break between positions 6538-6544, 6539- 6540 or 6543-6544 of human PDCD1. In another embodiment, the cell composition is characterized by increase (augmentation) of the relative level of the non-membrane bound PD-1 isoform transcript as compared to the full-length PD-1 isoform transcript. In another embodiment, said cells are T cells. In another embodiment, said cells further express any one of a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In another embodiment, said cells do not express a CAR or an engineered TCR.

In another embodiment, the cell composition is for use in treating cancer or for inducing or enhancing anti-tumor immunity in a subject in need thereof. In another embodiment, the subject is afflicted with a treatment-resistant tumor. In another embodiment, said subject is afflicted by a tumor characterized by PD-L1 over-expression. In another embodiment, said subject is afflicted by a PD-L1 non-expressing tumor.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A presents full length PD-1 (flPD-1) expression levels in human peripheral blood mononuclear cells (PBMC) following transduction with a vector encoding a PD-1 -specific sgRNA and a Blue Fluorescent Protein (BFP) marker, evaluated by flow cytometry. BFP+ denotes cells successfully transduced with the vector (fluorescent BFP positive cells), and BFP- denotes cells non-successfully transduced with said vector (non-fluorescent, BFP negative cells). FCS indicates forward scatter. Fig. IB presents soluble PD-1 (sPD-1) protein levels detected by ELISA in supernatants of PBMC following successful transduction with the PD-l-specific plasmid ("sgRNAl ") and of PBMC transduced with a plasmid encoding a control sgRNA (scrambled, nontargeting guide; Ctrl). Student t- test * p= 0.023.

Fig. 2A presents the fold-change in expression, compared to control cells, of PD-1 variants (flPD-1 and sPD-1) in Jurkat cells following transduction with the PD-l-specific vector (sgRNAl). Jurkat cells transduced with a construct encoding the non-targeting guide served as the control (Ctrl). Expression was evaluated using qPCR. Fig. 2B presents flPD-1 expression levels evaluated by flow cytometry in unstained Jurkat cells (unstained, vertical stripes), PD-1 knockout Jurkat cells (PD-1 KO, black line) and Jurkat cells following transduction with the constructs encoding the PD-1 -specif die (sgRNAl, dashed line) or non-specific guide and Ctrl, horizontal stripes). Fig. 2C presents flPD-1 expression levels evaluated by flow cytometry in unstained engineered Jurkat cells expressing a tumor- specific TCR (unstained, vertical strips), PD-1 knockout engineered Jurkat cells (PD-1 KO, black line) and engineered Jurkat cells following transduction with the constructs encoding the PD-l-specific (sgRNAl, dashed line) or non-specific guide (Ctrl, dashed line) and following co-culture with NY-ESO-1 peptide loaded T2 cells. Fig. 2D presents activation-induced IL-2 secretion determined by ELISA in Jurkat cells transduced with the non-targeting guide (Ctrl), PD-1 knockout Jurkat cells (PD-1 KO) and Jurkat cells following transduction with the PD-l-specific plasmid (sgRNAl). Anova test **<0.01, ****<0.001.

Fig. 3A illustrates normal T-cells expressing flPD-1 that are inhibited by PDL-1 expressed on cancer cells. Fig. 3B illustrates T cells secreting a soluble form of PD-1 following manipulation of PD-1 alternative splicing, resulting in PDL-1 blockage and improved T-cell killing capabilities.

FIG. 4 presents FACS analysis of cleaved caspase-3 positive melanoma 624 wild type (Mel 624) and melanoma 624 overexpressing PD-L1 (Mel 624- PD-L1) cells following co-culture with PBMCs activated and transduced with either both the melanoma- specific TCR and the gene editing system comprising the PD-1 isoform-specific sgRNA ("PD-1 splicing manipulation"). Two control groups: PBMCs transduced with a plasmid encoding a control sgRNA (scrambled, nontargeting guide; "SCR Control") or PBMC with a full knock out of the PD-1 gene ("PD-1 KO"). Cells were stained with a cell tracer marker DDAO and for cleaved caspase-3 as an indicator of early apoptosis.

Fig. 5A presents flPD-1 expression levels evaluated by flow cytometry in TIL 209 cells following electroporation with Ribonucleic Proteins (RNPs)-based constructs, with guides providing PD-1 knockout (“RNP PD-1 KO”), PD-1 isoform-specific gene editing (“RNP gRNAl”), or a non-targeting guide (“RNP Scr”). Fig. 5B presents sPD-1 expression levels in TIL 209 following the electroporation in the “RNP gRNAl”, “Scr RNP”, and “RNP PD-1 KO” groups. Additionally, an anti PD-L1 antibody “(atezo”) was added to each of the following TIL groups; “RNP Scr +atezo”, “RNP gRNAl+azeto”, “PD-1 KO+azeto”. The expression levels of sPD-1 were evaluated by ELISA. Fig. 5C presents FACS plots of the precent of cleaved caspase-3 positive Mel 624 and Mel 624- PD-L1 cells following co-culture with TILs introduces with either the "RNP gRNAl", "RNP SCR” or "RNP PD-1 KO". Cells were stained with a cell tracer marker DDAO and for cleaved caspase-3 as an indicator of early apoptosis.

Fig. 6A presents flPD-1 expression levels evaluated by flow cytometry in unstained TIL 209 cells (“unstained”), and TIL cells following transfection with the PD-1 isoform-specific gene editing RNPs having the targeting sequence of SEQ ID NO 3 (“RNP gRNAl”) or with the control non-targeting RNP (“RNP Scr”). Fig. 6B presents flPD-1 expression levels following transfection with RNPs having the targeting sequence of SEQ ID NO: 15 (“RNP gRNA4”). Fig. 6C presents flPD-1 expression levels following transfection with RNPs having the targeting sequence of SEQ ID NO: 17 (“RNP gRNA5”). Fig. 6D presents the mean fluorescence intensity (“MFI) of the flow cytometry curves as described in Figs. 6A-6C.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to nucleic acid agents modulating the expression of PD-1 isoforms, and methods of using same in immunotherapy and immunomodulation. Specifically, provided are nucleic acid molecules, including in particular gene-editing agents and antisense oligonucleotides (ASOs), nucleic acid constructs encoding them, and methods of using same. In some embodiments, nucleic acid agents and molecules of the invention may be used for inducing or enhancing splice switching in PD-1 expressing cells. The invention further relates to uses of the advantageous oligonucleotides, constructs and gene-editing systems in the preparation of cell compositions for immunotherapy, including in particular adoptive transfer cell compositions.

In some embodiments, the invention relates to a programmed cell death protein 1 (PD-1)- specific expression-modulating nucleic acid molecule, comprising 15-30 contiguous nucleotides that are specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 1 (TTTGTGCCCTTCCAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGC). According to a particularly advantageous embodiment, the nucleic acid molecule is specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2, as follows: TCCAGAGAGAAGGGCAGAAG (SEQ ID NO: 2).

In another embodiment the nucleic acid molecule has the nucleic acid sequence as set forth in SEQ ID NO: 3, as follows: CUUCUGCCCUUCUCUCUGGA (SEQ ID NO: 3). In another embodiment the nucleic acid molecule has the nucleic acid sequence as set forth in SEQ ID NO: 15, as follows: UUCUGCCCUUCUCUCUGGAA (SEQ ID NO: 15). In another embodiment the nucleic acid molecule has the nucleic acid sequence as set forth in SEQ ID NO: 17, as follows: GGCACUUCUGCCCUUCUCUC (SEQ ID NO: 17). In another embodiment the nucleic acid molecule is a synthetic guide RNA (gRNA). In another embodiment the nucleic acid molecule is a spliceswitching oligonucleotide. In another embodiment, the invention relates to a nucleic acid construct encoding the nucleic acid molecule.

It is to be understood, that when the nucleic acid sequence of a nucleic acid molecule of the invention is presented herein, both DNA and RNA sequences are included. For example, the sequence of a nucleic acid molecule having the nucleic acid sequence as set forth in SEQ ID NO: 3 may be either CTTCTGCCCTTCTCTCTGGA or CUUCUGCCCUUCUCUCUGGA, depending on the context (e.g. DNA or RNA, respectively) in which the molecule is used.

In other embodiments, the invention provides an engineered, non-naturally occurring gene-editing system (e.g., a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene-editing system), comprising or encoding: (i) the gRNA and (ii) an RNA-guided DNA endonuclease enzyme.

In other embodiments there is provided an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene-editing system, comprising, or encoding: (i) a synthetic guide RNA (gRNA) molecule and (ii) an RNA-guided DNA endonuclease enzyme, wherein the gRNA molecule is a programmed cell death protein 1 (PD-l)-specific expressionmodulating nucleic acid molecule that downregulates the expression of the full-length PD-1 isoform and up-regulates the expression of a non-membrane bound PD-1 isoform, and wherein said molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2.

In other embodiments, there are provided pharmaceutical compositions, which comprise as the active ingredient: the nucleic acid molecule; a nucleic acid construct encoding same; the CRISPR gene-editing system; a vector encoding the nucleic acid molecule or system; or a host cell encoding same, wherein each possibility represents a separate embodiment of the invention. Optionally, the pharmaceutical composition further contains a pharmaceutically acceptable carrier, excipient or diluent.

In another embodiment, the pharmaceutical composition is for use in the treatment of cancer. In another embodiment, the pharmaceutical composition is for use in inducing or enhancing anti-tumor immunity. In another embodiment, the pharmaceutical composition is for use in inducing or enhancing splice switching in PD-1 expressing cells. In another embodiment, the pharmaceutical composition is for use in preparing a cell composition adapted for immunotherapy. In a particular embodiment said pharmaceutical composition is for use in preparing a T cell composition adapted for adoptive transfer immunotherapy. In another particular embodiment said pharmaceutical composition is for use in preparing a natural killer (NK) cell composition adapted for adoptive transfer immunotherapy. In another embodiment, the invention relates to a cell composition adapted for immunotherapy that is prepared by the method.

In other embodiment, there is provided a method of inducing or enhancing splice switching in PD-1 expressing cells, comprising introducing into the cells an engineered, non- naturally occurring CRISPR gene-editing system, comprising, or encoding: (i) a synthetic gRNA molecule and (ii) an RNA-guided DNA endonuclease enzyme, wherein said gRNA molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2. In various embodiments, said gRNA molecule has a targeting sequence (that is specifically hybridizable with the nucleic acid target) selected from the group consisting of: SEQ ID NOs: 3, 15 and 17, or is a sgRNA molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 16 and 18, wherein each possibility represents a separate embodiment of the invention.

In other embodiments, there is provided a method of preparing a cell composition for immunotherapy, comprising the step of introducing into a leukocyte population, an engineered, non-naturally occurring CRISPR gene-editing system, in an amount and under conditions suitable for inducing or enhancing splice switching in the leukocyte population, wherein the system comprises, or encodes: (i) a synthetic gRNA molecule and (ii) an RNA-guided DNA endonuclease enzyme, and wherein said gRNA molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2. In various embodiments, said gRNA molecule has a targeting sequence (that is specifically hybridizable with the nucleic acid target) selected from the group consisting of: SEQ ID NOs: 3, 15 and 17, or is a sgRNA molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 16 and 18, wherein each possibility represents a separate embodiment of the invention.

In other embodiments there is provided a cell composition for immunotherapy, prepared by the method comprising the step of introducing into a leukocyte population, an engineered, non- naturally occurring CRISPR gene-editing system, in an amount and under conditions suitable for inducing or enhancing splice switching in the leukocyte population, wherein the system comprises, or encodes: (i) a synthetic gRNA molecule and (ii) an RNA-guided DNA endonuclease enzyme, and wherein said gRNA molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2. In another embodiment there is provided a cell composition for immunotherapy, prepared by the method as disclosed herein.

These and other embodiments of the invention will be described in greater detail below.

Nucleic acid agents

The nucleic acid agents designed according to the teachings of the present invention (also referred to in some embodiments as nucleic acid molecules) can be generated according to any nucleic acid synthesis method known in the art, including both enzymatic syntheses or solid-phase syntheses, as well as using recombinant methods well known in the art.

Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the nucleic acid agents is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example: Sambrook, J. and Russell, D. W. (2001), "Molecular Cloning: A Laboratory Manual"; Ausubel, R. M. et al., eds. (1994, 1989), "Current Protocols in Molecular Biology," Volumes I- III, John Wiley & Sons, Baltimore, Maryland; Perbal, B. (1988), "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York; utilizing solid-phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting, and purification by, for example, an automated trityl-on method or HPLC. It will be appreciated that nucleic acid agents of the present invention can be also generated using an expression vector as is further described herein.

The nucleic acid molecules of the invention, and in particular when used as exogenously- administered oligonucleotides, are typically derivatized by one or more backbone and/or sugar chemical modifications. For example, antisense oligonucleotides (ASOs) of the invention intended for inducing splice- switching in vivo advantageously contain modifications conferring resistance to nuclease-induced enzymatic degradation, and in particular to RNase H, that may degrade the pre-mRNA-ASO complex. Advantageously, oligonucleotides according to embodiments of the invention contain one or more 2' sugar modifications. For example, said modifications may advantageously be selected from the group consisting of 2’-O-Methyl (2'-O- Me), 2’-O-methoxyethyl (2'-M0E), and combinations thereof. Additionally or alternatively, the modifications may contain nucleic acid analogs comprising e.g. a 2'-O, 4'-C methylene bridge, such as locked nucleic acids (LNA). Additionally or alternatively, oligonucleotides of embodiments of the invention may also contain phosphorothiate (PS) backbone modification, phosphorodiamidate morpholinos (PMOs), and/or other modifications that may provide improved in vivo properties such as stability, tolerability, and bio-distribution. As used herein, "uniformly modified" or “fully modified” refers to an oligonucleotide or a region of nucleotides wherein essentially each nucleoside is a sugar modified nucleoside having uniform modification. As used herein, a "nucleoside" is a base-sugar combination and "nucleotides" are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.

As used herein, a nucleoside with a modified sugar residue is any nucleoside wherein the ribose sugar of the nucleoside has been substituted with a chemically modified sugar moiety. In the context of the present disclosure, the chemically modified sugar moieties include, but are not limited to, 2'-O-methoxyethyl, 2'-fluoro, 2'-dimethylaminooxyethoxy, 2'- dimethylaminoethoxy ethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, 2'-carbamate, 2'- aminooxy, 2'-acetamido and locked nucleic acid.

Modified nucleic acid (e.g. oligonucleotide) backbones may include, for example: phosphorothioates; chiral phosphorothioates; phosphorodithioates; phosphotriesters; aminoalkyl phosphotriesters; methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates; phosphinates; phosphoramidates, including 3'-amino phosphoramidate and aminoalkylphosphoramidates; thionophosphoramidates; thionoalkylphosphonates; thionoalkylphosphotriesters; and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms of the above modifications can also be used.

Alternatively, modified oligonucleotide/nucleic acid molecule backbones that do not include a phosphorus atom therein have backbones that are formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

Other nucleic acid agents which may be used according to the present invention are those modified in both sugar and the internucleoside linkage, i.e., the backbone of the nucleotide units is replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example of such an oligonucleotide mimetic includes a peptide nucleic acid (PNA). A PNA oligonucleotide refers to an oligonucleotide where the sugar- backbone is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The bases are retained and are bound directly or indirectly to aza-nitrogen atoms of the amide portion of the backbone.

Nucleic acid agents of the present invention may also include base modifications or substitutions. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). "Modified" bases include but are not limited to other synthetic and natural bases, such as: 5- methylcytosine (5-me-C); 5 -hydroxymethyl cytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyl uracil and cytosine; 6-azo uracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8- substituted adenines and guanines; 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5- substituted uracils and cytosines; 7-methylguanine and 7 -methyladenine; 8-azaguanine and 8- azaadenine; 7-deazaguanine and 7-deazaadenine; and 3 -deazaguanine and 3 -deazaadenine. Additional modified bases include those disclosed in: U.S. Pat. No. 3,687,808; Kroschwitz, J. I., ed. (1990), pages 858-859; Englisch et al. (1991); and Sanghvi (1993). Such modified bases are particularly useful for increasing the binding affinity of the oligonucleotides of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6-substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2°C, and may be advantageous even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

The term "hybridization" as used herein is generally used to mean hybridization of nucleic acids at appropriate conditions of stringency as would be readily evident to those skilled in the art depending upon the nature of the probe sequence (e.g. gRNA) and target sequences. Conditions of hybridization and washing are well known in the art, and the adjustment of conditions depending upon the desired stringency by varying incubation time, temperature and/or ionic strength of the solution are readily accomplished. See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989. The choice of conditions is dictated by the length of the sequences being hybridized, in particular, the length of the probe sequence, the relative G-C content of the nucleic acids and the amount of mismatches to be permitted. Low stringency conditions are preferred when partial hybridization between strands that have lesser degrees of complementarity is desired. When perfect or near perfect complementarity is desired, high stringency conditions are preferred. For typical high stringency conditions, the hybridization solution contains 6X S.S.C., 0.01 M EDTA, IX Denhardfs solution and 0.5% SOS. Hybridization is carried out at about 68°C for about 3 to 4 hours for fragments of cloned DNA and for about 12 to about 16 hours for total eukaryotic DNA. For lower stringencies the temperature of hybridization is reduced to about 42°C below the melting temperature (TM) of the duplex. The TM is known to be a function of the G-C content and duplex length as well as the ionic strength of the solution.

As used herein, "complementary" refers to a nucleic acid molecule that can form hydrogen bond(s) with another nucleic acid molecule by either traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotides. In reference to the nucleic acid molecules (e.g. antisense oligonucleotides or gRNA) of the present disclosure, the binding free energy for the oligonucleotide with its complementary sequence is sufficient to allow the relevant function of the antisense oligonucleotide or gRNA to proceed and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of ex vivo or in vivo therapeutic treatment. Determination of binding free energies for nucleic acid molecules is well known in the art. Thus, "complementary" (or "specifically hybridizable" or "capable of hybridizing in a selective manner") are terms that indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between a nucleic acid molecule (e.g. antisense oligonucleotide or gRNA) and a gene, pre-mRNA or mRNA target. It is understood in the art that a nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable. That is, two or more nucleic acid molecules may be less than fully complementary.

Complementarity is indicated by a percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively. Percent complementarity of a nucleic acid molecule with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art. Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman. "Perfectly" or "fully" complementary nucleic acid molecules means those in which all the contiguous residues of a first nucleic acid molecule will hydrogen bond with the same number of contiguous residues in a second nucleic acid molecule, wherein the nucleic acid molecules either both have the same number of nucleotides (i.e., have the same length) or the two molecules have different lengths.

Thus, in the context of the present disclosure, a nucleic acid sequence specifically hybridizable with a nucleic acid target as set forth herein is sufficiently complementary to its designated target such that, upon administration (e.g. to a PD-1 expressing cell) under physiological conditions, will bind to its target and not to other sequences within the cell's genome or transcriptome. For example, the invention relates in advantageous embodiments thereof to a PD-l-specific expression-modulating nucleic acid molecule, comprising 15-30 contiguous nucleotides which are specifically hybridizable with a nucleic acid target selected from the group consisting of SEQ ID NOs: 1 or 2. Thus, the molecule in question is sufficiently complementary to its designated target (SEQ ID NOs: 1 or 2) such that, upon administration to a PD-1 expressing cell under physiological conditions, it will bind to its target in a specific manner. As used herein, the terms "precursor mRNA" or "pre-mRNA" refer to an immature single strand of messenger ribonucleic acid (mRNA) that contains one or more intervening sequence(s) (introns). Pre-mRNA is transcribed by an RNA polymerase from a DNA template in the cell nucleus and is comprised of alternating sequences of introns and coding regions (exons). Once a pre-mRNA has been completely processed by the splicing out of introns and joining of exons, it is referred to as "messenger RNA" or "mRNA," which is an RNA that is completely devoid of intron sequences. Eukaryotic pre-mRNAs exist only transiently before being fully processed into mRNA. When a pre-mRNA has been properly processed to an mRNA sequence, it is exported out of the nucleus and eventually translated into a protein by ribosomes in the cytoplasm.

As used herein, the terms "splicing" and "(pre-)mRNA processing" refer to the modification of a pre-mRNA following transcription, in which introns are removed and exons are joined. Pre-mRNA splicing involves two sequential biochemical reactions. Both reactions involve the spliceosomal transesterification between RNA nucleotides. In a first reaction, the 2'-OH of a specific branch-point nucleotide within an intron, which is defined during spliceosome assembly, performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site forming a lariat intermediate. In a second reaction, the 3'-OH of the released 5' exon performs a nucleophilic attack at the last nucleotide of the intron at the 3' splice site thus joining the exons and releasing the intron lariat. Pre-mRNA splicing is regulated by intronic silencer sequence (ISS), exonic silencer sequences (ESS) and terminal stem loop (TSL) sequences. In other embodiments, splicing may be regulated by intronic enhancers (IES) and exonic enhancers (EES).

As used herein, "modulation of splicing" or "splice switching" refers to altering the processing of a pre-mRNA transcript such that there is an increase or decrease of one or more splice products, or a change in the ratio of two or more splice products. Modulation of splicing can also refer to altering the processing of a pre-mRNA transcript such that a spliced mRNA molecule contains either a different combination of exons as a result of exon skipping or exon inclusion, a deletion in one or more exons, or additional sequence not normally found in the spliced mRNA (e.g., intron sequence). In some embodiments (for example when the manipulation is performed using a gene-editing system as disclosed herein), the term further refers to alterations in the corresponding gene sequence, such that processing of its pre-mRNA transcript and the resulting gene products are altered as compared to the cell prior to manipulation (or to an equivalent non-manipulated cell).

In another embodiment said nucleic acid molecule is a single- stranded RNA molecule. In another embodiment said nucleic acid molecule is derivatized by one or more backbone and/or sugar chemical modifications. In another embodiment said nucleic acid molecule comprises one or more 2' sugar modifications. In another embodiment said modifications are selected from the group consisting of 2’-O-Methyl (2'-0-Me), 2’-O-methoxyethyl (2'-M0E), and combinations thereof, wherein each possibility represents a separate embodiment of the invention. In another embodiment said nucleic acid molecule is fully derivatized by 2'-0-Me or 2'-M0E.

The invention in embodiments thereof relates to PD-1 -specific expression-modulating nucleic acid molecules. In another embodiment said molecule downregulates the expression of the full-length PD-1 isoform and up-regulates the expression of a non-membrane bound PD-1 isoform. In another embodiment said molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2. As disclosed herein, the nucleic acid molecule is typically a splice- switching oligonucleotide, or is amenable for use in a gene editing system. For example, the nucleic acid molecule may be a synthetic guide RNA (gRNA) molecule (e.g. sgRNA).

As used herein, a PD-1 -specific expression-modulating nucleic acid molecule is a polynucleotide or oligonucleotide, which modulates the expression of at least one PD-1 isoform when introduced to (administered to or expressed in) a target cell. Further, the modulation (up- regulation and/or down-regulation) is PD-1 -specific, facilitated by specific hybridization of the nucleic acid molecule to a PDCD1 gene or PD-1 transcript in the target cell, such that expression of non-related genes is not affected. It is to be understood, however, that as a result of the modulation in the PD-1 isoform expression, downstream genes that are regulated by PD-1 isoforms may be subsequently affected indirectly. As the expression modulation induced by nucleic acid molecules of the invention results in a modified expression pattern of PD-1 isoforms, in which the expression of the at least one isoform is up-regulated or downregulated independently of the expression of other PD-1 isoforms (differential modulation), the nucleic acid molecules of the invention are further referred to herein as "PD-1 isoform-specific expression-modulating nucleic acid molecules".

The phrase PD-1 -specific expression-modulating nucleic acid molecule refers in particular to expression-modulating oligonucleotides that alter or facilitate modulation of PD-1 splicing and splice products in the target cell, such as PD-l-specific SSOs and gRNAs. In a particularly advantageous embodiment, said PD-l-specific expression-modulating nucleic acid molecule is a PD-l-specific gRNA, which differentially modulates the expression of PD-1 isoforms when introduced to a target cell in combination with an RNA-guided nuclease. Thus, the manipulation typically results in an augmented ratio of the non-membrane bound PD-1 isoform (sPD-1) to the full-length PD-1 isoform. Preferably, said gRNA down-regulates the expression of the full-length PD-1 isoform and up-regulates the expression of anon-membrane bound PD-1 isoform, e.g. when introduced to human T cells.

For example, the sPD-1 to flPDl transcript ratio in native lymphocytes is typically lower than 1. According to exemplary embodiments, expression-modulating nucleic acid molecules of the invention facilitate augmentation of the sPD-1 to flPDl transcript ratio to be higher than 1, e.g. at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, or 2, and typically about 1-5, 1.1-4, 1.5-3, 2-3.5, 2-3 or 1.8-2.8, wherein each possibility represents a separate embodiment of the invention.

As used herein, the term "gRNA" refers to a piece of RNAs that function as guides for RNA- or DNA-targeting enzymes, which they form complexes with. For example, gRNA can be designed to be used for targeted editing, such as with CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9. The targeting specificity of CRISPR-Cas9 is determined by a short sequence (e.g. 20-nt) at the 5' end of the gRNA. The desired target sequence must precede the protospacer adjacent motif (PAM). After base pairing of the gRNA to the target, Cas9 mediates a double strand break about 3-nt upstream of PAM. A single gRNA typically encodes a combination of the target homologous sequence (crRNA) and the scaffold RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The tracrRNA provide the "stem loop" structure required for binding to the endonuclease enzyme. The gRNA/Cas9 complex is recruited to the target sequence by the basepairing between the gRNA sequence and the complement genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break.

Advantageously, the present invention provides a gRNA comprising a PD-1 -specific expression-modulating nucleic acid molecule of 15-30 contiguous nucleotides in length, specifically hybridizable with a nucleic acid target of SEQ ID NO: 1. In another embodiment the present invention provides a gRNA comprising a PD-l-specific expression-modulating nucleic acid molecule of 15-30 contiguous nucleotides in length, specifically hybridizable with a nucleic acid target of SEQ ID NO: 2. In another embodiment the nucleic acid molecule (or the targeting sequence in case of a gRNA) is at least 90%, at least 95% or at least 98% complementary to the nucleic acid target. In another embodiment said nucleic acid molecule is 18-22 nucleotides in length. In another embodiment the crRNA sequence of said nucleic acid molecule is 18-22 nucleotides in length. In another embodiment said nucleic acid molecule has the nucleic acid sequence as set forth in SEQ ID NO: 3. In another embodiment said crRNA is of the nucleic acid sequence as set forth in SEQ ID NO: 3. In other embodiments, the gRNA is a sgRNA having the nucleic acid sequence as set forth in SEQ ID NO: 4.

As explained above gRNA molecules may comprise, in addition to the targeting sequence (crRNA), additional sequences such as the tracrRNA (in the case of sgRNA molecules) or additional residues that facilitate annealing of the crRNA to the tracrRNA. It is therefore to be understood that in a gRNA as disclosed herein, the targeting sequence is specifically hybridizable with the target sequence. For example, a PD-l-specific gRNA may have at least 15, 16, 17, 18, 19 and up to 20 contiguous nucleotides which are specifically hybridizable with the nucleic acid target of SEQ ID NO: 2. Each possibility represents a separate embodiment of the invention. Similarly, in such molecules the degree of complementarity is calculated based on the targeting sequence, and the non-targeting sequences such as tracrRNA are not included in the calculation. Thus, for example, advantageous gRNAs of the invention are characterized by a targeting sequence that is at least 90%, at least 95% or at least 98% complementary to the nucleic acid target of SEQ ID NO: 2. According to particular embodiments, the targeting sequence of said gRNA is selected from the group consisting of SEQ ID NOs: 3, 15 and 17, as described in Example Table 1 below. In a particular embodiment the targeting sequence of said gRNA is as set forth in SEQ ID NO: 3. In a particular embodiment the targeting sequence of said gRNA is as set forth in SEQ ID NO: 15. In a particular embodiment the targeting sequence of said gRNA is as set forth in SEQ ID NO: 17. In another embodiment said sgRNA has a nucleic acid sequence as set forth in any one of SEQ ID NOs: 4, 16 and 18. Each possibility represents a separate embodiment of the invention.

In other embodiments, additional specific gRNA targets within SEQ ID NO: 1 may be determined using a variety of publicly available bioinformatic tools including the CHOPCHOP algorithm, Broad Institute GPP, CasOFFinder, CRISPOR, Deskgen, etc. Methods for evaluating the efficacy of the nucleic acid agents and modulators include, for example, DNA sequencing, PCR, RT-PCR, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis.

In some embodiments, the present invention provides an engineered, non-naturally occurring CRISPR gene-editing system, comprising, or encoding: (i) a gRNA according to the invention and (ii) an RNA-guided DNA endonuclease enzyme. In another embodiments, the geneediting system comprises a first nucleic acid sequence encoding the gRNA, and a second nucleic acid sequence encoding the RNA-guided DNA endonuclease enzyme. In another embodiment said endonuclease enzyme is Cas9 or Cpf 1.

The terms "CRISPR gene-editing system", “CRISPR system,” “Cas system” or “CRISPR/Cas system” refer to a set of molecules comprising or providing an RNA-guided nuclease and a guide RNA (gRNA) molecule that together are necessary and sufficient to direct and effect modification of nucleic acid at a target sequence by the RNA-guided nuclease. In one embodiment, a CRISPR system comprises a guide RNA molecule and a Cas protein, e.g., a Cas9 protein. Such systems comprising a Cas9 or modified Cas9 molecule are referred to herein as “Cas9 systems” or “CRISPR/Cas9 systems.” In one example, the guide RNA molecule and Cas molecule may be complexed, to form a ribonuclear protein (RNP) complex. In some embodiments, the components of a CRISPR system may include a nucleic acid(s) (e.g., a vector) encoding one or more components of the system, a component(s) in protein form, or a combination thereof. In another embodiment, the gRNA is provided in the form of a sgRNA.

Naturally-occurring CRISPR systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. In contradistinction, the invention in embodiments thereof refers to engineered, non-naturally occurring CRISPR gene-editing systems, which are artificial systems (produced by synthetic or recombinant methods) that are not found as such (including its nuclease and gRNA components) in a single naturally-occurring cell or organism. These systems may comprise a wild type or naturally-occurring CRISPR nuclease; alternatively, the CRISPR nuclease can be engineered to have improved specificity, altered PAM specificity, decreased off-target effects, increased stability, and the like.

In another embodiment there is provided a nucleic acid construct encoding a PD-1 -specific expression-modulating nucleic acid molecule of 15-30 contiguous nucleotides in length, specifically hybridizable with a nucleic acid target selected from the group consisting of SEQ ID NOs: 1 or 2. In another embodiment there is provided a nucleic acid construct encoding a nucleic acid molecule as disclosed herein. In another embodiment the construct is an expression vector capable of expressing said nucleic acid molecule in human T cells. In another embodiment there is provided a host cell comprising the construct.

In some embodiments e.g. when the targeting gRNA is not covalently linked to the tracrRNA, or for in vivo administration, the gRNA molecule can be stabilized using modifications. According to certain embodiments, the gRNA is modified at the 5’ end. In certain other embodiments, said gRNA comprises one or more stability-enhancing modifications (such as 2’- O-methyl modifications) that are known in the art to be implemented with no loss of editing efficiency.

The term “construct” as used herein includes a nucleic acid sequence encoding a gene product such as a nucleic acid molecule according to the present invention, the nucleic acid sequence being operably linked to a promoter and optionally other transcription regulation sequences.

The phrase “operably linked” refers to linking a nucleic acid sequence to a transcription control sequence in a manner such that the molecule is able to be expressed when transfected (i.e., transformed, transduced, infected or transfected) into a host cell. Transcription control sequences are sequences, which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Exemplary suitable transcription control sequences include those that function in animal, bacteria, helminth, yeast and insect cells. The constructs of the invention comprise mammalian transcription control sequences, preferably human regulatory sequences, and, optionally and additionally, other regulatory sequences.

In another embodiment, the construct is an expression vector capable of expressing said nucleic acid molecule in human T cells. The term “expression vector” refers to a nucleic acid construct that directs expression of an RNA or polypeptide gene product from sequences linked to the promoters and/or other transcriptional regulatory sequences on the construct. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, transcription, transcript processing, translation and protein folding, modification and processing. The nucleic acid construct of methods and compositions of the present invention is, in another embodiment, a eukaryotic expression vector. In another embodiment, the nucleic acid construct is a plasmid. In another embodiment, the nucleic acid construct is any other type of expression vector capable of mediating expression in a target cell such as an immune cell or a cancer cell. Each possibility represents a separate embodiment of the present invention.

In an expression vector capable of expressing a nucleic acid molecule in human T cells, expression control sequences operatively linked to the gene product to be expressed include promoters and other elements that are active in human T cells. The promoter can be of genomic origin or synthetically generated. A variety of promoters for use in T cells are well-known in the art. The promoter can be constitutive or inducible, where induction is associated with the specific cell type or a specific level of activation or maturation, for example. Alternatively, a number of well-known viral promoters are also suitable. Promoters of interest include but are not limited to the P-actin promoter, SV40 early and late promoters, immunoglobulin promoter, human cytomegalovirus promoter, retrovirus promoter, and the Friend spleen focus-forming virus promoter. The promoters may or may not be associated with enhancers, wherein the enhancers may be naturally associated with the particular promoter or associated with a different promoter.

Exemplary strong well-characterized promoters for inducing expression in human T cells include the human immediate early cytomegalovirus (CMV) promoter, a strong ubiquitous classically used viral promoter; Murine stem cell virus (MSCV) retroviral LTR promoter, previously described in T cells to mimic transcription regulation of y-retroviral vector; Human phosphoglycerate kinase (PGK), an endogenous housekeeping promoter showed to sustain a moderate and stable expression level; Beta-2-Microglobulin (P2m) promoter, an ubiquitous and constitutive promoter especially strong in immune cells; Human elongation factor 1 alpha (EFla), a strong ubiquitous constitutive promoter classically used for stable gene transfer, and other promoters such as hPGK and RPBSA promoters. Such promoters may conveniently be used for ex vivo manipulation of T cells, where a constitutive expression of the manipulation is needed, for example in the preparation of ACT compositions. In some embodiments, e.g. for in vivo gene editing, T cell-specific and/or inducible promoters are preferred. In some embodiments, the promoter is a T cell-specific promoter, e.g. a CD3, CD4 or CD8 promoter. Exemplary inducible promoters initiating expression of a gene product upon lymphocyte or T cell activation include NFAT, API, and NR4A promoters.

The construct may also comprise other regulatory sequences or selectable markers, as known in the art. Other than containing the necessary elements for the transcription of the inserted coding sequence, the expression construct of the present invention can also include sequences engineered to improve stability, production, purification, yield or toxicity of the expressed oligonucleotide.

In another embodiment, a nucleic acid molecule (e.g. gRNA), construct, system or vector as disclosed herein (collectively referred to herein as PD-1 -modulating agents), modulates the expression of at least one PD-1 isoform in T-cells. Thus, said agent downregulates and/or up- regulates the expression of PD-1 isoforms when introduced to (administered or expressed in) T- cells (e.g. primary human T cells).

In another embodiment, said PD-1 -modulating agent enhances anti-tumor immunity of said cells. Thus, said agent induces or significantly enhances the tumor-specific immune response in the T cells to which it is introduced. Without wishing to be bound by a specific theory or mechanism of action, it is disclosed herein that differential modulation of PD-1 isoform expression induced by splice- switching, following administration of gene editing systems in accordance with the invention, is associated with enhanced tumor killing, tumor- specific cytotoxicity, and tumor-induced T cell activation (manifested e.g. by enhanced IL-2 secretion). In another embodiment, said agent modulates the expression of at least one PD-1 isoform in T- cells to thereby enhance anti-tumor immunity of said cells.

In various embodiments, the invention relates to a nucleic acid molecule, construct, system or vector as disclosed herein, which modulates the expression of at least one PD-1 isoform in T- cells and enhances anti-tumor immunity of said cells. In some embodiments, said nucleic acid molecule, construct, system or vector modulates the expression of at least one PD-1 isoform in T- cells to thereby enhance anti-tumor immunity of said cells.

In various embodiments, the invention relates to a nucleic acid molecule, construct, system or vector as disclosed herein, which downregulates the expression of the full-length PD-1 isoform and up-regulates the expression of a non-membrane bound PD-1 isoform. In another embodiment, the nucleic acid molecule, construct, system or vector downregulates the expression of the full- length PD-1 isoform transcript and up-regulates the expression of a 5' truncated transcript of a PD-1 isoform, lacking at least the transmembrane domain (due to a 5' proximal deletion, caused by e.g. alternative splicing). In a particular embodiment, the transcript of the 5' truncated PD-1 isoform lacks exon 3. In another embodiment said isoform lacks exon 3 and substantially retains the remaining exons characteristic of full-length PD-1 (including in particular exon 2). In another embodiment said 5' truncated PD-1 isoform encodes a soluble (non-membrane bound) PD-1 protein (sPD-1). In another embodiment, the nucleic acid molecule, construct, system or vector downregulates the expression of the full-length PD-1 isoform transcript and up-regulates the expression of an alternative transcript of a PD-1 isoform lacking at least a part of the transmembrane domain. In another embodiment, the transcript of the alternatively spliced PD-1 isoform lacks exon 3. In another embodiment said isoform lacks exon 3 and substantially retains the remaining exons characteristic of full-length PD-1. In another embodiment said PD-1 isoform encodes a sPD-1 protein. In another embodiment, nucleic acid molecule, construct, system or vector as disclosed herein, which downregulates the expression of the full-length PD-1 isoform and up-regulates the expression of a sPD-1 isoform.

The term "down-regulation" (or downregulation) is used in the context of gene expression, and refers to a significant decrease in the amount of the target gene product with respect to a reference control. The term "up-regulation" (or upregulation), when used to describe gene expression, refers to a significant increase in the amount of said gene product with respect to the reference control. Thus, advantageous PD-1 -modulating agents as disclosed herein, when introduced to a target cell (e.g. T cell), downregulate the expression of the full-length PD-1 isoform (flPD-1) and up-regulate the expression of a non-membrane bound PD-1 isoform (sPD- 1), such that the level of the flPD-1 isoform (measured as mRNA or polypeptide) is significantly reduced compared to its control level prior to introduction (in the same cell or in a corresponding non-modulated control cell), and the level of the sPD-1 isoform is significantly enhanced compared to the control. For example, a downregulated PD-1 isoform mRNA may have a greater than 1.5-fold decrease in expression level, e.g. a decrease of 1.5-2.5-fold or about twofold in the case of full- length PD-1. An upregulated PD-1 isoform mRNA may have a greater than 1.5-fold increase in its expression level, e.g. an enhancement of 2.5-3.5-fold or about in the case of soluble PD-1.

Full-length human PD-1 is a 288-amino acid protein derived from the human PDCD1 gene. Full-length PD-1 comprises an extracellular N-terminal domain which is IgV-like, a transmembrane domain and an intracellular domain containing an immunoreceptor tyrosine-based inhibitory (ITIM) motif and an immunoreceptor tyrosine-based switch (ITSM) motif. The term "full-length PD-1" (or flPD-1) refers to a PD-1 polypeptide comprising the aforementioned motifs and domains. A representative amino acid sequence of full-length human PD-1 is provided in GenBank as accession number NM_005018.3, and is set forth in SEQ ID NOs: 21 and 22 (transcript and polypeptide, respectively), as follows:

GCTCACCTCCGCCTGAGCAGTGGAGAAGGCGGCACTCTGGTGGGGCTGCTCCAGGCA TGCAGATCCCACAGGCG CCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGAC TCCCCAGACAGGCCC TGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACC TTCACCTGCAGCTTCT CCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGG ACAAGCTGGCCGCCT TCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCA ACGGGCGTGACTTCC ACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCT CCCTGGCCCCCAAGG CGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGC CCACAGCCCACCC CAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGG CCTGCTGGGCAGCCT GGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGG AGCCAGGCGCACCG GCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGC TGGATTTCCAGTGGC GAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCA TTGTCTTTCCTAGCG GAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGC CACTGAGGCCTGAG GATGGACACTGCTCTTGGCCCCTCTGACCGGCTTCCTTGGCCACCAGTGTTCTGCAGACC CTCCACCATGAGCCCG GGTCAGCGCATTTCCTCAGGAGAAGCAGGCAGGGTGCAGGCCATTGCAGGCCGTCCAGGG GCTGAGCTGCCTGG GGGCGACCGGGGCTCCAGCCTGCACCTGCACCAGGCACAGCCCCACCACAGGACTCATGT CTCAATGCCCACAGT GAGCCCAGGCAGCAGGTGTCACCGTCCCCTACAGGGAGGGCCAGATGCAGTCACTGCTTC AGGTCCTGCCAGCAC AGAGCTGCCTGCGTCCAGCTCCCTGAATCTCTGCTGCTGCTGCTGCTGCTGCTGCTGCTG CCTGCGGCCCGGGGCT GAAGGCGCCGTGGCCCTGCCTGACGCCCCGGAGCCTCCTGCCTGAACTTGGGGGCTGGTT GGAGATGGCCTTGG AGCAGCCAAGGTGCCCCTGGCAGTGGCATCCCGAAACGCCCTGGACGCAGGGCCCAAGAC TGGGCACAGGAGTG GGAGGTACATGGGGCTGGGGACTCCCCAGGAGTTATCTGCTCCCTGCAGGCCTAGAGAAG TTTCAGGGAAGGTC AGAAGAGCTCCTGGCTGTGGTGGGCAGGGCAGGAAACCCCTCCACCTTTACACATGCCCA GGCAGCACCTCAGGC CCTTTGTGGGGCAGGGAAGCTGAGGCAGTAAGCGGGCAGGCAGAGCTGGAGGCCTTTCAG GCCCAGCCAGCACT CTGGCCTCCTGCCGCCGCATTCCACCCCAGCCCCTCACACCACTCGGGAGAGGGACATCC TACGGTCCCAAGGTCA GGAGGGCAGGGCTGGGGTTGACTCAGGCCCCTCCCAGCTGTGGCCACCTGGGTGTTGGGA GGGCAGAAGTGCA GGCACCTAGGGCCCCCCATGTGCCCACCCTGGGAGCTCTCCTTGGAACCCATTCCTGAAA TTATTTAAAGGGGTTG GCCGGGCTCCCACCAGGGCCTGGGTGGGAAGGTACAGGCGTTCCCCCGGGGCCTAGTACC CCCGCCGTGGCCTA TCCACTCCTCACATCCACACACTGCACCCCCACTCCTGGGGCAGGGCCACCAGCATCCAG GCGGCCAGCAGGCACC TGAGTGGCTGGGACAAGGGATCCCCCTTCCCTGTGGTTCTATTATATTATAATTATAATT AAATATGAGAGCATGCT AA (human flPD-1 transcript, SEQ ID NO: 21);

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS NTSESFVLNWYRIVISPSNQ. TDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQ.I KESLRAELRVTERRAEVPTA HPSPSPRPAGQ.FQ.TLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQ.PLKE DPSAVPVFSVDYGELDFQ.WRE KTPEPPVPCVPEQ.TEYATIVFPSGMGTSSPARRGSADGPRSAQ.PLRPEDGHCSWPL (human flPD-1, SEQ ID NO: 22).

Thus, for example, in SEQ ID NO: 21, positions 57-125 correspond to the signal peptide, positions 159-491 correspond to the IgV-like extracellular N-terminal domain, and positions 567- 629 correspond to the transmembrane domain; in SEQ ID NO: 22, positions 1-23 correspond to the signal peptide, positions 35-145 correspond to the IgV-like extracellular N-terminal domain, and positions 171-191 correspond to the transmembrane domain.

Since flPD-1 comprises a functional transmembrane domain, it may be anchored to the plasma membrane and displayed on the cell surface (either constitutively or induced upon activation or other physiological stimuli). In contradistinction, non-membrane bound PD-1 isoforms are not anchorable to the plasma membrane due to a deletion or mutation at the transmembrane domain. The term "non-membrane bound PD-1" encompasses naturally occurring splice variants and synthetic (engineered) PD-1 isoforms as disclosed herein. Non-membrane bound PD-1 polypeptides are typically secreted from the cell due to the lack of the anchoring function. Such secreted isoforms may also be referred to as soluble PD-1 polypeptides.

For example, as disclosed and demonstrated herein, lymphocytes manipulated by gene editing systems in accordance with the invention are characterized by up-regulation of a soluble PD-1 isoform (sPD-1) in which positions 493-648 with respect to the mRNA transcript of SEQ ID NO: 21 (corresponding to positions 146-197 of the polypeptide of SEQ ID NO: 22) have been deleted. Accordingly, a non-membrane-bound human PD-1 isoform in accordance with the invention may have the following sequence:

GCTCACCTCCGCCTGAGCAGTGGAGAAGGCGGCACTCTGGTGGGGCTGCTCCAGGCA TGCAGATCCCACAGGCG CCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGAC TCCCCAGACAGGCC CTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCAC CTTCACCTGCAGCTTC TCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACG GACAAGCTGGCCGC CTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCC CAACGGGCGTGACTT CCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCAT CTCCCTGGCCCCCA AGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGGGACAATAGGAGCCA GGCGCACCGGCC

AGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGC TGGATTTCCAGTGGCGA GAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATT GTCTTTCCTAGCGGA ATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCA CTGAGGCCTGAGGA TGGACACTGCTCTTGGCCCCTCTGACCGGCTTCCTTGGCCACCAGTGTTCTGCAGACCCT CCACCATGAGCCCGGG TCAGCGCATTTCCTCAGGAGAAGCAGGCAGGGTGCAGGCCATTGCAGGCCGTCCAGGGGC TGAGCTGCCTGGG

GGCGACCGGGGCTCCAGCCTGCACCTGCACCAGGCACAGCCCCACCACAGGACTCAT GTCTCAATGCCCACAGTG AGCCCAGGCAGCAGGTGTCACCGTCCCCTACAGGGAGGGCCAGATGCAGTCACTGCTTCA GGTCCTGCCAGCAC AGAGCTGCCTGCGTCCAGCTCCCTGAATCTCTGCTGCTGCTGCTGCTGCTGCTGCTGCTG CCTGCGGCCCGGGGC TGAAGGCGCCGTGGCCCTGCCTGACGCCCCGGAGCCTCCTGCCTGAACTTGGGGGCTGGT TGGAGATGGCCTTG GAGCAGCCAAGGTGCCCCTGGCAGTGGCATCCCGAAACGCCCTGGACGCAGGGCCCAAGA CTGGGCACAGGAG

TGGGAGGTACATGGGGCTGGGGACTCCCCAGGAGTTATCTGCTCCCTGCAGGCCTAG AGAAGTTTCAGGGAAGG TCAGAAGAGCTCCTGGCTGTGGTGGGCAGGGCAGGAAACCCCTCCACCTTTACACATGCC CAGGCAGCACCTCA GGCCCTTTGTGGGGCAGGGAAGCTGAGGCAGTAAGCGGGCAGGCAGAGCTGGAGGCCTTT CAGGCCCAGCCAG CACTCTGGCCTCCTGCCGCCGCATTCCACCCCAGCCCCTCACACCACTCGGGAGAGGGAC ATCCTACGGTCCCAA GGTCAGGAGGGCAGGGCTGGGGTTGACTCAGGCCCCTCCCAGCTGTGGCCACCTGGGTGT TGGGAGGGCAGAA

GTGCAGGCACCTAGGGCCCCCCATGTGCCCACCCTGGGAGCTCTCCTTGGAACCCAT TCCTGAAATTATTTAAAG GGGTTGGCCGGGCTCCCACCAGGGCCTGGGTGGGAAGGTACAGGCGTTCCCCCGGGGCCT AGTACCCCCGCCGT GGCCTATCCACTCCTCACATCCACACACTGCACCCCCACTCCTGGGGCAGGGCCACCAGC ATCCAGGCGGCCAGC AGGCACCTGAGTGGCTGGGACAAGGGATCCCCCTTCCCTGTGGTTCTATTATATTATAAT TATAATTAAATATGAG

AGCATGCTAA (human sPD-1 transcript, SEQ ID NO: 23); and

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS NTSESFVLNWYRMSPSNQ. TDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQ.I KESLRAELRVTGTIGARRTG QPLKEDPSAVPVFSVDYGELDFQ.WREKTPEPPVPCVPEQ.TEYATIVFPSGI\/1GTSS PARRGSADGPRSAQ.PLRPEDGHC SWPL (human sPD-1, SEQ ID NO: 24).

In another embodiment the invention relates to host cells comprising or expressing the nucleic acid molecules and systems of the invention. Various suitable prokaryotic and eukaryotic host cells with suitable expression vectors are known in the art, including, but not limited to animal cells (including mammalian cells, e.g. human cells, Chinese hamster ovary cells (CHO) or COS cells), bacterial cells, plant cells, yeast cells and insect cells. According to certain advantageous embodiments, the host cell is a human cell, e.g. a leukocyte population as disclosed herein. For example, the host cell may be e.g. human T cells including, but not limited to tumor infiltrating leukocytes (TIL), tumor- specific T cell clones, and genetically modified T cells. In another embodiment, said host cell is formulated as a T cell containing population that expresses a chimeric antigen receptor (CAR). In another embodiment said host cell does not express a CAR or a chimeric TCR. In another embodiment, said host cell is a T cell containing population that expresses an autologous engineered T cell receptor (TCR). In another specific embodiment said host cell is a T cell composition adapted for adoptive transfer immunotherapy as disclosed herein. In other embodiments, the host cell may be selected from T cells (e.g. CD8 ⁺ , CD4 ⁺ or gamma- delta CD3 ⁺ T cells), B cells, NK cells, antigen presenting cells and tumor-associated macrophages.

In another embodiment the host cells are derived from PBMC. In another specific embodiment said host cell is formulated as a cell composition adapted for immunotherapy as disclosed herein. Each possibility represents a separate embodiment of the invention.

Table 1 below identifies the designations and SEQ ID NOs. of certain nucleic acid molecules employed in the construction of RNA guides and evaluated in the context of gene editing systems in the Examples section below. Pharmaceutical compositions

According to other embodiments, the nucleic acid agents, such as the oligonucleotides, nucleic acid molecules, gene editing systems and constructs described herein, or the host cells encoding them, are formulated in the form of a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier, excipient or diluent, as detailed below. In another embodiment, there is provided a pharmaceutical composition comprising an engineered, non-naturally occurring CRISPR gene-editing system, comprising, or encoding: (i) a synthetic gRNA molecule and (ii) an RNA-guided DNA endonuclease enzyme, wherein said gRNA molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2. In various embodiments, said gRNA molecule has a targeting sequence (that is specifically hybridizable with the nucleic acid target) selected from the group consisting of: SEQ ID NOs: 3, 15 and 17, or is a sgRNA molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 16 and 18, wherein each possibility represents a separate embodiment of the invention.

Pharmaceutical compositions comprising the nucleic acid molecules, systems and vectors described herein may comprise any pharmaceutically acceptable salts, esters, or salts of such esters, or any other functional chemical equivalent which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the nucleic acid molecules, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

The term "prodrug" indicates a therapeutic agent that is prepared in an inactive or less active form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes, chemicals, and/or conditions. In particular, prodrug versions of the oligonucleotides are prepared as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods disclosed in WO 93/24510 or WO 94/26764. Prodrugs can also include nucleic acid molecules wherein one or both ends comprise nucleotides that are cleaved (e.g., by incorporating phosphodiester backbone linkages at the ends) to produce the active compound.

The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For example, sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans. The nucleic acid molecules described herein may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds.

The present disclosure also includes pharmaceutical compositions and formulations which include the nucleic acid molecules described herein. The pharmaceutical compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. In a particular embodiment, administration is intramuscular or intravenous. In another embodiment, intratumoral administration is contemplated.

The pharmaceutical formulations, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product (e.g., into a specific particle size for delivery). In a particular embodiment, the pharmaceutical formulations are prepared for intramuscular administration in an appropriate solvent, e.g., water or normal saline, possibly in a sterile formulation, with carriers or other agents.

A "pharmaceutical carrier" or "excipient" can be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal and are known in the art. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.

The nucleic acid molecules described herein may be in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Nucleic acid molecule compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. Suspensions may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The present disclosure also includes nucleic acid molecules compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co., A.R. Gennaro edit., 1985). For example, preservatives and stabilizers can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

Pharmaceutical compositions of this disclosure can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxy ethylene sorbitan monooleate.

The nucleic acid molecules of this disclosure may be administered to a patient by any standard means, with or without stabilizers, buffers, or the like, to form a composition suitable for treatment. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. Thus, the nucleic acid molecules of the present disclosure may be administered in any form, for example intramuscular or by local, systemic, or intrathecal injection.

This disclosure also features the use of nucleic acid molecules compositions comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of nucleic acid molecule in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated nucleic acid molecule. Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of nucleic acid molecules, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (PCT Publication No. WO 96/10391; WO 96/10390; and WO 96/10392). Long-circulating liposomes are also likely to protect nucleic acid molecules from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

Typical dosage amounts of nucleic acid molecules in pharmaceutical formulations may range from about 0.05 to 1000 mg/kg body weight, and in particular from about 5 to 500 mg/kg body weight. In one embodiment of the invention and/or embodiments thereof, the dosage amount is from about 50 to 300 mg/kg body weight once in 2 weeks, or once or twice a week, or any frequency required to achieve therapeutic effect.

The dosage administered will, of course, vary depending on the use and known factors such as the pharmacodynamic characteristics of the active ingredient; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. The recipient may be any type of mammal, but is preferably a human. In one embodiment of the invention and/or embodiments thereof, dosage forms (compositions) of the inventive pharmaceutical composition may contain about 1 microgram to 50,000 micrograms of active ingredient per unit, and in particular, from about 10 to 10,000 micrograms of active ingredient per unit. For intravenous delivery, a unit dose of the pharmaceutical formulation will generally contain from 0.5 to 500 micrograms per kg body weight and preferably will contain from 5 to 300 micrograms, in particular 10, 15, 20, 30, 40, 50, 100, 200, or 300 micrograms per kg body weight (pg/kg body weight) of the nucleic acid molecule. Preferred intravenous dosage ranges from 10 ng to 2000 pg, preferably 3 to 300 pg, more preferably 10 to 100 pg of compound per kg of body weight. In one particular embodiment, it should be recognized that the dosage can be raised or lowered based on individual patient response. It will be appreciated that the actual amounts of nucleic acid molecule used will vary according to the specific nucleic acid molecule being utilized, the particular compositions formulated, the mode of application, and the particular site of administration.

In a particular embodiment, nucleic acid molecules of the invention may be delivered in vivo alone or in association with a vector (expression vector or delivery vector). In its broadest sense, a "vector" is any vehicle (e.g. nucleic acid construct as disclosed herein) capable of facilitating the transfer of the nucleic acid molecule of the invention to the cells. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, naked plasmids, non-viral delivery systems (electroporation, sonoporation, cationic transfection agents, liposomes, etc.), phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the nucleic acid molecule nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: RNA or DNA viruses such as a retrovirus (as for example moloney murine leukemia virus and lentiviral derived vectors), harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno- associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors according to the invention include adenoviruses and adeno- associated (AAV) viruses, which are DNA viruses that have already been approved for human use in gene therapy. 12 different AAV serotypes (AAV1 to 12) are known, each with different tissue tropisms. Recombinant AAV are derived from the dependent parvovirus AAV. The adeno- associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Another preferred example of viral vectors according to the invention include lentivirus, which is a genus of retrovirus. Retroviruses contain an RNA genome that is converted to DNA in the transduced cell by reverse transcriptase. Advantageously, lentiviral infection is characterized by high-efficiency infection of dividing and non-dividing cells, long-term stable expression of a transgene, and low immunogenicity. Lentiviral vectors have become particularly attractive for clinical applications due to their ability to more efficiently transduce non-proliferating or slowly proliferating cells. Lentiviral vectors have also been used to deliver components of gene editing, such as guide RNAs for the CRISPR-Cas9 system. Third-generation self-inactivating (SIN) lentiviral vectors, in particular, have demonstrated safety when used to transfer genes into both stem cells and T cells. No cases of insertional oncogenesis have been reported with a natural HIV or gene therapy using lentiviral vectors.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a gene product from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by, intranasal sprays or drops, rectal suppository and orally. Preferably, said DNA plasmid is injected intramuscular, or intravenous. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleates and microencap sulation .

In a preferred embodiment of the invention and/or embodiments thereof, a nucleic acid (in particular antisense oligonucleotide or gRNA) sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters. In another embodiment of the invention and/or embodiments thereof, the vector may code for more than one ASO or gRNA. In other exemplary embodiments, gene editing systems of the inventions can be delivered in-vivo using e.g. AAV, lentiviral vectors, lipid nanoparticles, polymer nanoparticles, peptide nanoparticles, inorganic nanoparticles, DNA nanostructures or exosome-based delivery systems known in the art (e.g. as disclosed by Duan et al, 2021, Frontiers in Genetics, vol. 12, #673286).

In yet other embodiments, gene editing systems of the inventions intended for clinical applications may advantageously be used in the form of RNPs, e.g. as disclosed and exemplified herein.

In some embodiments, there is provided a pharmaceutical composition comprising a nucleic acid construct encoding an oligonucleotide or nucleic acid molecule as disclosed herein, e.g. an expression vector capable of expressing said oligonucleotide or nucleic acid molecule in human T cells. In other embodiments there is provided a pharmaceutical composition comprising an oligonucleotide or nucleic acid molecule as disclosed herein. In other embodiments there is provided a pharmaceutical composition comprising a host cell as disclosed herein (e.g. a population of human T cells including, but not limited to an adoptive transfer cell composition as disclosed herein). In yet other embodiments, the pharmaceutical composition comprises an engineered, non-naturally occurring CRISPR gene-editing system as disclosed herein.

Subjects and methods

In various embodiments, the invention relates to compositions and methods for the treatment of cancer, for inducing or enhancing anti-tumor immunity and/or for inducing or enhancing splice switching, wherein each possibility represents a separate embodiment of the invention. According to particularly advantageous embodiments, the compositions and methods are used in the treatment or subjects afflicted with, or at risk for developing, treatment resistance. Thus, the invention in embodiments thereof relates to the treatment of new patient populations, not hitherto considered amenable for treatment by PD-1- or PD-L1 targeted therapies such as immune checkpoint inhibitors and gene editing systems.

In various embodiments, the methods comprise administering to a subject, or expressing in cells of the subject, one or more PD-1 -specific expression-modulating nucleic acid molecules as disclosed herein. In other embodiments, the methods comprise administering to a subject, or expressing in cells of the subject, one or more engineered, non-naturally occurring CRISPR geneediting systems comprising or encoding the PD-1 -specific expression-modulating nucleic acid molecules as disclosed herein. In yet other embodiments, the nucleic acid molecule or gene editing system are administered in the form of a host cell modified by said nucleic acid molecule or gene editing system, e.g. a cell composition as disclosed herein. In another embodiment there is provided a pharmaceutical composition comprising the nucleic acid molecule, construct or host cell as disclosed herein, and optionally a pharmaceutically acceptable carrier, excipient or diluent, for use in the treatment of cancer, for inducing or enhancing anti-tumor immunity and/or for inducing or enhancing splice switching, wherein each possibility represents a separate embodiment of the invention.

In another embodiment, the methods comprise introduction or administration of an engineered, non-naturally occurring CRISPR gene-editing system, comprising, or encoding: (i) a synthetic gRNA molecule and (ii) an RNA-guided DNA endonuclease enzyme, wherein said gRNA molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2. In various embodiments, said gRNA molecule has a targeting sequence (that is specifically hybridizable with the nucleic acid target) selected from the group consisting of: SEQ ID NOs: 3, 15 and 17, or is a sgRNA molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 16 and 18, wherein each possibility represents a separate embodiment of the invention.

In some embodiments, the subject to be treated by the compositions and methods of the invention is afflicted with cancer, or at risk for developing cancer (e.g. afflicted with a pre- cancerous lesion or diagnosed with a condition associated with high risk for tumor formation). In another embodiment said subject has been diagnosed with cancer. Advantageously, said subject is a human subject. As used herein, the terms "cancer" and "tumor" are used interchangeably and include both solid tumors and hematopoietic tumors.

In another embodiment, the cancer is a solid tumor. In various embodiments, the cancer is selected from the group consisting of melanoma and other skin cancers, renal cell carcinoma, lung cancer, breast cancer, colon cancer and other cancers of the intestinal tract, bladder cancer, pancreatic cancer, prostate cancer, blood and bone marrow cancer and head and neck cancer, wherein each possibility represents a separate embodiment of the invention. In other embodiments, the cancer may be e.g. urinary tract cancer, gynecological cancer, head and neck carcinoma, primary brain tumor, bladder cancer, ovarian cancer, prostate cancer, cervical cancer, bone malignancies or connective and soft tissue tumors. In another embodiment the cancer or tumor is melanoma, renal cell carcinoma, lung cancer, breast cancer, head and neck cancer, bladder cancer, urothelial cancer, pancreatic cancer and cervical cancer. In another embodiment, the tumor is selected from endometrial tumors and germ cell tumors. In another embodiment the cancer is metastatic. In another embodiment the compositions and methods are used for preventing or delaying the formation of tumor metastasis. In another embodiment, said tumor is characterized by DNA repair deficiency. In another embodiment said tumor is a microsatellite stable (MSS) tumor. In another embodiment said tumor is characterized by a mismatch repair deficiency (microsatellite instability-high tumor). For example, MSS colorectal cancer is an exemplary tumor considered resistant to immunotherapy, as will be discussed in greater detail below.

In some embodiments, the cancer or tumor is a PD-Ll-expressing tumor. In another embodiment, the tumor is characterized by PD-L1 over-expression. For example, without limitations, PD-L1 over-expressing tumors (exhibiting elevated PDL-1 levels) include without limitation solid tumors such as melanoma, non-small cell lung cancer (NSCLC), bladder cancer, renal cell carcinoma, head and neck tumors, and cervical tumors. In yet other advantageous embodiments, said tumor is a PD-L1 -non-expressing tumor, expressing PD-L1 on less than 1% of the cells.

In some embodiments, the tumor to be treated is a tumor amenable for treatment with an adoptive cell therapy. In some cases (for example, in lung cancer and bladder cancer), said tumors are characterized by PD-L1 up-regulation. In other cases (e.g. in melanoma and renal tumors), the tumor may either be PD-Ll-expressing or PD-L1 -non-expressing. In yet other embodiments, the tumor may be a treatment-resistant tumor, not amenable for other therapies. In another embodiment, said tumor is characterized by DNA repair deficiency. Each possibility represents a separate embodiment of the invention.

In another embodiment the subject is further treated by a cancer immunotherapy. In another embodiment the method further comprises administering to the subject a cancer immunotherapy. In a particular embodiment the cancer immunotherapy is a T-cell mediated immunotherapy (directed at inducing, enhancing or otherwise modulating the activity of T cells in the subject). Exemplary T-cell mediated immunotherapies include, but are not limited to, T cell-modulating cytokines, immune checkpoint inhibitors and cell composition immunotherapies (e.g. adoptive transfer cell compositions) as disclosed herein.

For example, without limitation, the subject is further treated by one or more cytokines (e.g. IL-2, IL-7, IL-12, IL-15, and IL-21). In other exemplary embodiments, co-treatment with immune checkpoint inhibitors (e.g. directed to PD-1, CTLA4, TIGIT, TIM3, PD-L1, 41BB, FAS, GITR, BTLA, ICOS, 0X40, LAG-3, GLECTIN 9, CD96, VISTA, H-VEM, P-SELECTIN, or PCGL), is contemplated. In another embodiment, said tumor is resistant to treatment by one or more of the above-mentioned cytokines or immune checkpoint inhibitors, wherein each possibility represents a separate embodiment of the invention. In another embodiment, said tumor is an immunotherapy resistant tumor, not amenable for treatment with immunotherapies such as cytokines and immune checkpoint inhibitors. In a particular embodiment said tumor is resistant to treatment with PD-1 antagonists and inhibitors. In another particular embodiment, said tumor is resistant to treatment with PD-L1 antagonists and inhibitors. In another embodiment, the nucleic acid agents of the invention are used as monotherapy.

The term "resistance" refers to the feature of cancer (or tumor) not responding to a given treatment. A tumor can be resistant to a particular therapy already at the initiation of treatment (primary resistance). Alternatively, a tumor can develop resistance during the course of treatment (acquired resistance, also referred to as secondary resistance). The resistance to therapy is commonly understood as unsatisfactory effectiveness of treatment usually resulting in disease progression. In the case of acquired resistance, the resistance can also be manifested as a decrease in the amount of tumor regression at the same dose or an increase in the dose necessary for the same amount of tumor regression. For example, in the case of resistance to immune checkpoint inhibitors, the term “resistant” refers to a tumor that fails, or has failed, to respond adequately in a favorable manner in terms of tumor shrinkage or duration of stabilization or shrinkage in response to treatment with an immune checkpoint inhibitor for a time of greater than 3 months or more.

The term "amenable for treatment" refers to a tumor predicted or determined to respond favorably to the treatment. Amenability for treatment can be determined by the treating physician or oncologist by assessing clinical response to the treatment (or to a similarly acting treatment of the same category) or predicted by the skilled artisan by assessing cell viability or apoptosisinducing activity after bringing a suitable anti-cancer agent in contact with a tumor or tumor sample originating from a subject. The skilled person is aware of the existence of standard assays to determine treatment amenability or screen for resistant cancers, such as MTT assays, ATP- measurements and/or apoptosis-assays such as TUNEL, Cytochrome C release or Cleaved Caspase-3 assays.

In a particular embodiment said tumor is resistant to treatment (or considered non- amenable) with PD-1 -directed immunotherapies, namely immunotherapies targeted to PD-1 or a ligand thereof. Such therapies include for example PD-1 and/or PD-L1 checkpoint inhibitors, and cell therapies such as adoptive cell compositions genetically manipulated or edited to inactivate or eliminate expression of PD-1 ("PD-1 knock-out"). For example, many tumors are intrinsically resistant to PD-l-directed immunotherapies despite expressing PD-L1. For instance, about 60% of melanomas are intrinsically resistant to PD-1 and/or PD-E1 checkpoint inhibitors. As disclosed herein, gene editing systems in accordance with embodiments of the invention may be used for producing adoptive cell compositions effective in treating both PD-Ll-expressing and PD-L1 non-expressing tumors (e.g. melanomas), with a substantially reduced incidence of resistance. Thus, these systems and compositions may be advantageously used even in the treatment of patient populations not hitherto considered to be amenable for treatment with PD-1 and/or PD-L1 checkpoint inhibitors.

In another embodiment, the pharmaceutical compositions of the invention are for use in inducing or enhancing splice switching in PD-1 expressing cells. In another embodiment, there is provided a method for inducing or enhancing splice switching in PD-1 expressing cells, comprising introducing into the cells one or more PD-1 -specific expression-modulating nucleic acid molecules of the invention or a system as disclosed herein. In another embodiment the method comprises introducing into the cells an engineered, non-naturally occurring CRISPR gene-editing system comprising, or encoding: (i) a gRNA as disclosed herein and (ii) an RNA- guided DNA endonuclease enzyme. In another embodiment the gRNA comprises 15-30 contiguous nucleotides that are specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2, e.g. a gRNA comprising a targeting sequence selected from the group consisting of SEQ ID NOs: 3, 15 and 17. In some embodiments, the system is introduced into the cells in vitro or ex vivo. In yet other embodiments, said system is introduced into the cells in vivo. Each possibility represents a separate embodiment of the invention.

As used herein, inducing or enhancing splice switching refers to exerting a significant or otherwise detectable increase or decrease of one or more splice products associated with splice switching in the treated cell as compared to the cell prior to manipulation (or to an equivalent nonmanipulated cell). For example, inducing or enhancing splice switching may include enhancing the expression of a non-membrane bound PD-1 isoform (measured as mRNA transcript levels) by at least 1.5-fold and typically at least 2-, 2.5-, 3- or 3.5-fold, e.g. 2.5-3.5-fold or about 3-fold. In other examples, inducing or enhancing splice switching may include reducing the expression of the full-length PD-1 transcript by at least 1.2-fold and typically about 1.5-2.5-fold or about 2-fold. According to other exemplary embodiments, inducing or enhancing splice switching refers to augmentation of the sPD-1 to flPDl transcript ratio to be higher than 1, e.g. at least 1.1 or 1.3, and typically 1.1-5, 1.5-3 or 2.5-3.5. Inducing or enhancing splice switching as referred to herein is also manifested by altered functional properties, e.g. enhanced tumor- specific immunity and cytotoxicity, as disclosed herein. In some embodiments, the full-length PD-1 transcript is not completely downregulated, and the cells still maintain a significant or otherwise detectable level of the full-length transcript (as opposed, for example, to cells subjected to PD-1 knockout). For example, as demonstrated herein, a cell resulting from induction or enhancement of splice- switching may be distinguishable from a cell subjected to PD-1 knockout by gene editing by significantly enhanced levels of both sPD-1 and flPD-1 as compared to the corresponding PD-1 knockout cell.

As used herein, the term PD-1 expressing cells is used to denote cells expressing at least one PD-1 gene product or that can be induced to express at least one gene product (e.g. upon activation or in the presence of cytokines or other physiological stimuli). In a particular embodiment, the cells are characterized by PD-1 surface expression (e.g. of full-length PD-1). Exemplary PD-1 expressing cells are T cells, natural killer (NK) and B lymphocytes, macrophages, dendritic cells (DCs) and monocytes. Exemplary PDL-1 expressing cells are T cells, NK cells, macrophages, myeloid DCs, B cells, epithelial cells, and vascular endothelial cells that can be induced to express PD-L1, e.g. upon IFN-y stimulation, and PD-L1 expressing tumors including, but not limited to myeloid tumors, lung tumors and bladder tumors.

The terms "expressing" or "positive" are well-known in the art and denote that a gene product is expressed in an amount detectable by an approach known in the art. A protein can be detected by use of immunological assay, for example, ELISA, immuno staining, or flow cytometry, e.g. using a labeled antibody. A transcript can be detected by use of a method of amplifying and/or detecting nucleic acid, for example, RT-PCR, microarray, biochip, or RNAseq.

The term “not expressing” or “negative” means that the expression level of a protein or gene of a cell is less than a lower limit of detection by all or any of the known techniques. The respective techniques may have different detection lower limits of expression of the protein or transcript. When referring to a cell population (e.g. a tumor) as not expressing a gene product, it is meant that said gene product is expressed by a non- substantial portion of the cells, such that a function associated with the gene product is not effectively provided. For example, in the case of PD-L1, non-expressing tumors are tumors characterized by less than 1% surface expression. Such tumors are also considered resistant to (or not amenable with) PD-1 -directed therapy.

As used herein, “over-expressing" a polypeptide refers to expression of a polypeptide at higher-than-normal levels compared to those observed with corresponding healthy control or “wild-type" cells or tissue. Exemplary cells over-expressing PD-L1 are cells derived from certain solid tumors such as melanoma, non-small cell lung cancer (NSCLC), bladder cancer, renal cell carcinoma, head and neck tumors, and cervical tumors. For example, in the case of NSCLC, tumors characterized by over 50% PD-L1 expression are considered PD-L1 over-expressing tumors. Such tumors are typically considered amenable for PD-l-directed therapy (e.g. PD-1 or PD-L1 antagonists) as monotherapy. NSCLC tumors expressing PD-L1 on between 1% and 50% of the cells are considered PD-L1 expressing tumors. Such tumors are typically considered amenable for treatment with PD-l-directed therapy in combination with chemotherapy, whereas NSCLC tumors characterized by less than 1% PD-L1 expression are typically not considered amenable for PD-l-directed therapy.

Similarly, triple negative breast cancer, esophageal squamous cell carcinoma, and head and neck tumors are indicated for PD-l-directed therapy (either with or without chemotherapy). It is to be understood, that the specific threshold may be determined by the treating physician based on the characteristics of the specific tumor, patient and treatment. For example, gastric tumors are typically considered for PD-l-directed therapy if they express PD-L1 on 5-10% of the tumor cells, depending on the specific PD-l-directed therapy (e.g. PD-1 antagonist antibody) to be used.

In a particular embodiment, the subject is further treated with (or the method further comprises administration of) an adoptive cell therapy composition as disclosed herein (e.g. prepared by administering to, or expressing in, a T cell population, a nucleic acid molecule or system of the invention, in an amount and under conditions suitable for inducing or enhancing splice switching in the T cell population). In a particular embodiment, the subject is further treated with (or the method further comprises administration of) another cell composition immunotherapy, as disclosed herein.

Cell compositions and adoptive cell therapy

In some embodiments, the invention relates to cell compositions, including adoptive transfer cell compositions and other cell compositions amenable for immunotherapy, and to methods for their preparation. The term “cell composition” as used herein indicates a pharmaceutical composition that contains cells or cellular material as the active ingredient. Cell compositions typically contain pharmaceutically acceptable carriers, excipients or diluents, and optionally additional components other than cells such as culture medium or preservation liquid.

In another aspect, there is provided a cell composition prepared by a method comprising the step of administering to, or expressing in, a leukocyte population, a PD-1 -specific expressionmodulating nucleic acid molecule or a system as disclosed herein, in one embodiment, the system is an engineered, non-naturally occurring CRISPR gene-editing system, comprising, or encoding: (i) a synthetic gRNA molecule and (ii) an RNA-guided DNA endonuclease enzyme, wherein said gRNA molecule comprises 15-30 contiguous nucleotides specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2. In various embodiments, said gRNA molecule has a targeting sequence (that is specifically hybridizable with the nucleic acid target) selected from the group consisting of: SEQ ID NOs: 3, 15 and 17, or is a sgRNA molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 16 and 18, wherein each possibility represents a separate embodiment of the invention.

In another embodiment, administration is performed in an amount and under conditions suitable for inducing or enhancing splice switching in the leukocyte population. In other embodiments, the leukocyte population may be selected from PBMC, T cells (e.g. CD8 ⁺ , CD4 ⁺ and gamma-delta CD3 ⁺ T cells), B cells, NK cells, antigen presenting cells and tumor-associated macrophages (e.g. engineered to express CAR, an engineered TCR or other receptors or beneficial molecules). In another embodiment, said cells do not express a CAR or an engineered TCR. In other embodiments, the leukocyte population may be selected from antigen presenting cells (e.g. macrophages or dendritic cells) that present TAA or that were loaded with tumor lysate, useful as an anti-tumor vaccine. In another embodiment, said cells are selected from the group consisting of PBMC, tumor infiltrating leukocytes (TIL), tumor- specific T cell clones, genetically modified T cells, B cells, NK cells, antigen presenting cells and tumor-associated macrophages, macrophages and dendritic cells. Each possibility represents a separate embodiment of the invention.

For example, the nucleic acid molecule or system may be administered to APC (e.g. dendritic cells - DC) used for an anti-tumor vaccination protocol. According to an exemplary protocol, DC are generated by stimulation of monocytes isolated from PBMC with interleukin 4 (IL-4) and granulocyte-macrophage colony- stimulating factor (GM-CSF). DC-based vaccines may be generated by loading of tumor lysate or genetically engineering the cells to express TAA, by protocols known in the art.

A nucleic acid molecule of the invention (e.g. SSO or gRNA) or a system (e.g. in the form of an RNP) may be introduced into the cells e.g. by electroporation, using Nucleofector technology, (AMAXA), or by other transfection methods e.g. liposome-mediated transfer. The nucleic acid molecules may also be expressed in the cells following transfection or infection with a suitable construct (including, but not limited to viral vectors, e.g. AAV-based vectors, lentivirus vectors). Of further interest are delivery systems employing bio-conjugates for leukocyte targeting and/or cell penetration, e.g. aptamers, antibodies or sugars (for example. A'- acetylgalactosamine).

Various methods for ex vivo administration of gene editing systems may be employed to generate cell compositions of the invention. For example, gRNAs can be delivered ex-vivo into cells using viral transduction (e.g., as exemplified in Example 1), or by transfection of the gRNA using electroporation, e.g. as a CRISPR-Cas9 ribonucleoprotein (crRNP) complex as described by (Hultquist et al. 2019, Nature protocols vol. 14,1). In addition to direct administration of the CRISPR-Cas9 protein as described above, Cas9 may also be expressed in the target cells by the use of appropriate nucleic acid agents, e.g. following ex- vivo in mRNA electroporation, or by viral transduction (e.g. as exemplified in Example 3).

In another aspect, there is provided a cell composition prepared as described herein, suitable for use in immunotherapy. The terms "immunotherapy" and in particular "cancer immunotherapy" as used herein refers to the treatment or prevention of a disease or condition (e.g. in particular cancer) by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response. In the case of cancer immunotherapy, the modulation typically includes induction or enhancement of anti-tumor immunity or cytotoxicity.

As used herein, a "cell composition for immunotherapy" or "cell composition adapted for immunotherapy" is a cell composition formulated so as to be injected, grafted or implanted into a patient to induce or otherwise modulate an immune response in a subject. Exemplary cell compositions for immunotherapy include but are not limited to cell vaccines (e.g. cancer cell vaccines or DC vaccines) and adoptive cell transfer (ACT) compositions. In some embodiments, the cell composition for immunotherapy comprises live or attenuated immune cells (e.g. T cells) manipulated by the nucleic acids or systems of the invention and expanded ex vivo, as described herein.

In another aspect, there is provided a T cell composition prepared as described herein, suitable for adoptive transfer into a recipient subject in need thereof. As used herein, and unless otherwise specified, the term "adoptive transfer" refers to a form of passive immunotherapy where previously sensitized immunologic agents (e.g., cells or serum) are transferred to the recipients. The phrases “adoptive transfer immunotherapy”, “adoptive cell therapy” and “adoptive cell immunotherapy” are used interchangeably herein to denote a therapeutic or prophylactic regimen or modality, in which effector immunocompetent cells, such as the T cell compositions of the invention, are administered (adoptively transferred) to a subject in need thereof, to alleviate or ameliorate the development or symptoms of cancer or infectious diseases.

T lymphocytes (T cells) are one of a variety of distinct cell types involved in an immune response. The activity of T cells is regulated by antigen, presented to a T cell in the context of a major histocompatibility complex (MHC) molecule. The T cell receptor (TCR) then binds to the MHC-antigen complex. Once antigen is complexed to MHC, the MHC-antigen complex is bound by a specific TCR on a T cell, thereby altering the activity of that T cell. Proper activation of T lymphocytes by antigen-presenting cells requires stimulation not only of the TCR, but the combined and coordinated engagement of its co-receptors.

T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4 ⁺ T cells because they express the CD4 glycoprotein on their surfaces. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen- presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. Cytotoxic T cells (Tc cells, or CTLs) destroy virus-infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8 ⁺ T cells since they express the CD8 glycoprotein at their surfaces. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Regulatory T cells (T _re cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress autoreactive T cells that escaped the process of negative selection in the thymus.

The TCR is a complex of integral membrane proteins, wherein stimulation by specific MHC-presented antigen recognition and binding by the clono type- specific a/p heterodimer leads to activation of transcription and subsequent proliferation and effector functions (such as cytotoxic activity in CD8 ⁺ T cells and cytokine secretion in CD4 ⁺ T cells). This activation involves other subunits of the receptor complex as detailed below, that couple the extracellular ligand to downstream signaling pathways such as protein phosphorylation, the release of inositol phosphates and elevation of intracellular calcium levels.

The intracellular portions of the CD3 y, 5, 8, and subunits contain copies of a sequence motif termed ITAMs (immunoreceptor tyrosine-based activation motifs). ITAMs can serve as protein tyrosine kinase substrates and, after phosphorylation, as binding sites for SH2 domains of yet other kinases. The regulation and mechanism of the recruitment of protein kinases to the activated T cell receptor involves members of both the Syk family (ZAP-70) and Src family (Lek) of kinases.

TCR stimulation as detailed above may be antigen- specific or antigen non-specific (Polyclonal). Suitable antigen- specific TCR activators include antigens bound to MHC molecules, typically in the context of APCs. Polyclonal TCR activators are capable of initiating the signal transduction and transcriptional activation pathways associated with specific TCR engagement in the absence of specific antigens. Suitable polyclonal T cell activators include antibodies that bind and crosslink the T cell receptor/CD3 complex, e.g. subunits as described herein. Exemplary antibodies that crosslink the T cell receptor include the HIT3a, UCHT1 and OKT3 monoclonal antibodies. The stimulation is provided at an amount and under conditions as known in the art so as to induce the above-mentioned functional effects.

A cell composition (e.g. T cell composition) for adoptive transfer immunotherapy, also referred to herein as an "ACT composition", "adoptive transfer cell composition", " composition adapted for adoptive transfer immunotherapy" or "compositions for adoptive cell transfer", is an immune cell composition prepared and formulated for use in ACT. Suitable ACT compositions and methods for their preparation are described in further detail herein.

Typically, compositions for adoptive cell transfer are prepared by methods including activating a T cell population by a TCR stimulation, and expansion of the cells to obtain a therapeutically effective amount of effector T cells for administration. Such methods include but are not limited to, Rapid Expansion Protocols (REP).

In various embodiments, the TCR stimulation may be antigen non-specific (performed, for example, using antibodies specific to CD3 that activate the receptor upon binding, e.g. OKT3) or antigen- specific (using suitable antigen presenting cells and antigen). In the context of cancer treatment, antigen- specific stimulation typically employs stimulation to tumor-associated antigens. The term “tumor-associated antigen” (TAA) refers to any protein, peptide or antigen associated with (carried by, expressed by, produced by, secreted by, etc.) a tumor or tumor cell(s). Tumor-associated antigens may be (nearly) exclusively associated with a tumor or tumor cell(s) and not with healthy normal cells or may be over expressed (e.g., 2 times, 5 times, 10 times, 50 times, 100 times, 1000 times or more) in a tumor tissue or tumor cell(s) compared to healthy normal tissue or cells. More particularly, a TAA is an antigen capable of being presented (in processed form) by MHC determinants of the tumor cell. Hence, tumor-associated antigens are likely to be associated only with tumors or tumor cells expressing MHC molecules. Non-limitative examples of well-known TAA are MART-1, gplOO 209-217, gplOO 154-163, CSPG4, NY-ESO, MAGE-A1, Tyrosinase.

In some embodiments, one commonly used approach for stimulating proliferation, in particular of CD8 ⁺ T cells, is the incubation of T cells with soluble anti-CD3 antibody in the presence of Fc receptor-bearing accessory cells (feeder cells), an approach designated the REP. Antibody "presented" to T cells in this manner generates a more effective proliferative signal than soluble anti-CD3 alone or anti-CD3 immobilized on a plastic surface. In the treatment of cancer, adoptive cell therapy typically involves collecting T cells that are found within the tumor of the patient (referred to as tumor-infiltrating lymphocytes, TIL), which are encouraged to multiply ex vivo using high concentrations of IL-2, anti-CD3 and allo-reactive feeder cells. These T cells are then transferred back into the patient along with exogenous administration of IL-2 to further boost their anti-cancer activity.

Thus, according to certain additionally advantageous embodiments, activation and/or expansion (e.g. as part of a REP protocol) is performed in the presence of feeder cells. The term “feeder cells” generally refers to cells of one type that are co-cultured with cells of a second type, to provide an environment in which the cells of the second type can be maintained and proliferated. For the purpose of the present invention, this term specifically refers to Fc receptor-bearing accessory cells, which are typically allo-reactive with the T cell containing population to be propagated. In other words, the feeder cells need not be histocompatible with the T-cell containing population to be propagated, and in certain advantageous embodiments the two populations typically HLA-mismatched. A typical example of feeder cells is allogeneic normal donor peripheral blood mononuclear cells, PBMC. Typically and advantageously, the use of such feeder cells is performed in conjunction with antigen non-specific TCR stimulation, e.g. by incubation with antigen non-specific stimulating antibodies, as detailed herein.

In another embodiment, adoptive transfer T cell compositions are prepared with irradiated PBMC (incapable of proliferation) as feeder cells. For example, PBMC may conveniently be attenuated by irradiation by exposing the cells to 6000RAD. In another embodiment, adoptive transfer T cell compositions are prepared with artificial antigen presenting entities including antigen presenting cells and inert particles carrying antigens, to provide antigen- specific stimulation.

In various embodiments, T cell expansion may be performed for at least 5 and typically at least 6, 7, or 8 days. Typically, expansion is performed for up to about 16, 15, 14, 13, or 12 days, for example 5-15 days, e.g. 6-12 or more typically 8-15 days. In another embodiment, the population comprises CD8 ⁺ T cells. In another embodiment, the T cells are CD8 ⁺ T cells. In another embodiment, the cells are further genetically engineered or modified (e.g. to exert a desired antigen specificity).

The cell composition may comprise a T cell-containing population in an effective amount. For example, an amount effective for adoptive transfer immunotherapy is an amount sufficient to induce or enhance a beneficial immune response such as an anti-tumor response, e.g. 10 ⁶ to 10 ¹² cells. It is to be understood, that while cell preparations suitable for in vivo administration, particularly for human subjects, may contain pharmaceutically acceptable excipients or diluents, such preparations are sufficiently devoid of contamination by pathogens, toxins, pyrogens and any other biological and non-biological agents which are not recognized to be pharmaceutically acceptable. For example, without limitation, T cells for adoptive transfer immunotherapy may conveniently be suspended in an injection suitable buffer that contains sterile saline with 2% human albumin, and optionally IL-2 (e.g. 300IU/ml).

According to certain preferable embodiments, the cell composition is histocompatible with the subject to be treated (e.g. autologous cells or MHC Il-matched allogeneic cells). The term "histocompatibility" refers to the similarity of tissue between different individuals. The level of histocompatibility describes how well matched the patient and donor are. The major histocompatibility determinants are the human leukocyte antigens (HLA). HLA typing is performed between the potential donor and the potential recipient to determine how close an HLA match the two are. The term “histocompatible” as used herein refers to embodiments in which all six of the HLA antigens (2 A antigens, 2 B antigens and 2 DR antigens) are the same between the donor and the recipient.

However, in other embodiments, donors and recipients who are "mismatched" at two or more antigens, for example 5 of 6, or in other embodiments, 4 of 6 or 3 of 6 match, may be encompassed by certain embodiments of the invention, despite the donor and recipient not having a complete match. The term “substantially histocompatible” as used herein refers to embodiments in which five out of six of the HLA antigens are the same between the donor and the recipient.

In some embodiments of the methods of the invention, expanding the T cell population so as to obtain a T cell composition adapted for adoptive transfer immunotherapy comprising an effective amount of the resulting T cell population, is performed by a REP protocol.

Thus, in some embodiments, provided are methods for preparing a T cell composition adapted for adoptive transfer immunotherapy, comprising the step of administering to, or expressing in, a T cell population, a PD-1 specific expression-modulating nucleic acid molecule or a system as defined herein, in an amount and under conditions suitable for inducing or enhancing splice switching in the T cell population. In various embodiments, PD-l-specific expressionmodulating nucleic acid molecule is of 15-30 contiguous nucleotides in length, specifically hybridizable with a nucleic acid target selected from the group consisting of SEQ ID NOs: 1 or 2. In some embodiments said nucleic acid molecules are at least 90%, at least 95% or at least 98% complementary to the nucleic acid target. In a particular embodiment, said nucleic acid molecules are 18-22 nucleotides in length. In another embodiment said nucleic acid molecules have the nucleic acid sequence as set forth in SEQ ID NO: 3. In another embodiment said nucleic acid molecules have the nucleic acid sequence as set forth in SEQ ID NO: 15 or 17. In another embodiment said nucleic acid molecules are single- stranded RNA molecules. In another embodiment said nucleic acid molecules are derivatized by one or more backbone and/or sugar chemical modifications. In another embodiment said nucleic acid molecules comprise one or more 2' sugar modifications. In another embodiment said modifications are selected from the group consisting of 2’-O-Methyl (2'-O-Me), 2’-O-methoxyethyl (2'-MOE), and combinations thereof. In another embodiment said nucleic acid molecules are of SEQ ID NO: 3 and/or are fully derivatized by 2'-O-Me or 2'-MOE. In another embodiment said nucleic acid molecules are splice- switching nucleic acid molecules. In various embodiments, the system is an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene-editing system, comprising, or encoding: (i) a gRNA with a nucleic acid sequence of SEQ ID NO: 3 or 4 (or in other embodiments, SEQ ID NO: 15, 16, 17 or 18), and (ii) an RNA-guided DNA endonuclease enzyme. In some embodiments said gene-editing system, comprises a first nucleic acid sequence encoding the gRNA, and a second nucleic acid sequence encoding the RNA-guided DNA endonuclease enzyme. In another embodiment, said endonuclease is Cas9 or Cpfl. In another embodiment, the system is transferred/delivered to cells with a vector. In another embodiment, the vector is a viral vector.

In another embodiment, the method comprises: a. providing a T cell population (e.g. comprising CD8 ⁺ T cells), b. administering to, or expressing in the T cell population, one or more PD-l-specific expression-modulating nucleic acid molecule or system, in an amount and under conditions suitable for inducing or enhancing splice switching in said T cell population, and c. expanding said T cell population, so as to obtain a T cell composition adapted for adoptive transfer immunotherapy comprising an effective amount of the resulting T cell population.

According to various other specific embodiments, the composition is prepared according to specific protocols and parameters as disclosed herein, for example by the REP protocols described above. In another embodiment, step b may be performed by methods known in the art, for example the nucleic acid molecules may be administered to the cells e.g. by electroporation, using Nucleofector technology, (AMAXA), or by other transfection methods e.g. liposome- mediated transfer. The nucleic acid molecules may also be expressed in the cells following transfection or infection with a suitable construct (including, but not limited to viral vectors, e.g. AAV-based vectors, lentivirus vectors) encoding the nucleic acid molecules. Gene editing systems may conveniently be administered in the form of Ribonucleic Proteins (RNPs), or the vector encoding the gRNA and the endonuclease may be introduced separately, as disclosed and exemplified herein. Exemplary conditions for inducing or enhancing splice switching are provided throughout the specification and Examples herein.

According to exemplary embodiments, target cells (e.g. T cells) can be first transfected or transduced with a construct or vector expressing PD-1 isoform-specific sgRNA of the invention, and the endonuclease can then be introduced to sgRNA-expressing cells. For example, without limitation, electroporation of Cas9 protein into T cells transduced with a viral vector expressing a PD-1 isoform-specific sgRNA may be performed using a range of 1-100 pg Cas9 protein per 10 ⁶ transduced cells, e.g. 3-80, 5-50, 2-20 or about 10 pg Cas9 protein per 10 ⁶ transduced cells. Each possibility represents a separate embodiment of the invention.

In other exemplary embodiments when RNPs are used, PD-1 isoform-specific gRNA may be complexed with Cas9 protein at a ratio of gRNA to Cas9 protein of 1:1, 2:1, 3:1, 4:1, 5:1, or alternatively 1:2, 1:3, 1:4 or 1:5. The resulting RNPs may be administered to the target cells (e.g. T cells) at an amount of 50-1000 pmol of RNP to 1-50* 10 ⁶ cells, e.g. 70-500, 80-200 or 100-150 pmol RNPs per 10 ⁶ cells. Each possibility represents a separate embodiment of the invention.

In another embodiment, the cell composition (e.g. T cell composition) is for use in the treatment of cancer. In another embodiment the cell composition is for use in inducing or enhancing anti-tumor immunity. In another embodiment the tumor is a solid tumor. In another embodiment the tumor (or cancer) is selected from the group consisting of melanoma, renal cell carcinoma, lung cancer, breast cancer, head and neck cancer, bladder cancer, urothelial cancer, pancreatic cancer and cervical cancer. In another embodiment the cancer (or tumor) is characterized by PD-L1 over-expression.

In various embodiments, cell compositions of the invention are characterized by downregulated levels of full-length PD-1 and up-regulated levels of non-membrane bound PD-1. For example, without limitation, the step of administering to, or expressing in the T cell population, one or more PD-1 -specific expression-modulating nucleic acid molecules or systems, in an amount and under conditions suitable for inducing or enhancing splice switching in said T cell population, is performed so as to induce a decrease of 1.5-2.5-fold or about twofold of the full-length PD-1 transcript and an enhancement of 2.5-3.5-fold or about threefold of the soluble PD-1 transcript. In another embodiment, the step is performed so as to induce a decrease of at least about 23% and typically 23-100%, e.g. 25-40%, 35-70%, 40-90%, 30-80% or 25-95% of the full-length PD-1 polypeptide and an enhancement of at least 32% and typically about 32-55%, e.g. 35-45%, 30-40% or 30-53% of the soluble PD-1 polypeptide.

As disclosed herein, cell compositions of the invention, including in particular T cell compositions adapted for adoptive transfer immunotherapy, are characterized by unique structural and functional properties and are therefore differentiated from hitherto reported ACT compositions. Thus, in some embodiments, there is provided a T cell composition adapted for adoptive transfer immunotherapy, characterized by structural and functional properties as disclosed herein.

Structurally, cell compositions (e.g. ACT compositions) produced using gene editing systems of the invention are typically characterized by a deletion, addition or substitution mutation characteristic of a repaired double stranded break between positions 6538-6544 of the PDCD1 gene (positions counted from the most 5' nucleotide in exon 1 of NM_005018.3). Thus, disclosed herein are cell compositions comprising an indel mutation at a position as disclosed herein.

As used herein, the term “indel” collectively refers to a mutation in which at least one nucleotide is inserted and/or deleted at a locus of a DNA sequence. For example, when a double helix of DNA is cleaved by an RNA-guided nuclease, an indel may be introduced into the target sequence while the cleaved DNA is repaired by a homologous recombination or non-homologous end-joining (NHEJ) mechanism.

For example, as disclosed herein, gRNAs (e.g. sgRNAs) comprising the targeting sequence of SEQ ID NO: 3 result in the formation of a double stranded DNA break between positions 6539-6540 of human PDCD1 (positions counted from the most 5' nucleotide in exon 3 of NM_005018.3, namely between positions -3 to -2 relative to exon 3); gRNAs comprising the targeting sequence of SEQ ID NO: 15 result in the formation of a double stranded DNA break between positions 6538-6539 of human PDCD1 (between positions -4 to -3 relative to exon 3; and gRNAs comprising the targeting sequence of SEQ ID NO: 17 result in the formation of a double stranded DNA break between positions 6543-6544 of human PDCD1 (between positions 1 and 2 of exon 3).

Accordingly, advantageous cell compositions in accordance with the invention comprise a non-naturally occurring immune cell population characterized by a splice switching indel mutation as disclosed herein. In various embodiments, the mutation may be associated with NHEJ repair of a double- stranded DNA break between positions 6538-6544, 6539-6540 or 6543-6544 of human PDCD1. Each possibility represents a separate embodiment of the invention.

The phrase "splice switching indel mutation" as referred to herein indicates an indel mutation that induces or enhances splice- switching at the mutated gene. For example, a splice switching indel mutation at the PDCD1 (e.g. resulting from NHEJ repair of a double- stranded DNA break between positions 6538-6544, 6539-6540 or 6543-6544 of human PDCD1) induces or enhances exon 3 skipping in the PD-1 transcript, and augmentation of the relative level of the sPD-1 isoform transcript as compared to the flPD-1 isoform transcript.

In addition, cell compositions (e.g. ACT compositions) in accordance with the invention are characterized by an augmented (increased) ratio of the sPD-1 isoform to the full-length PD-1 isoform (as compared to corresponding non-manipulated control cells). As disclosed herein, cell compositions of the invention are typically characterized by an augmented sPD-1 to flPDl transcript ratio of at least 1 and typically higher, e.g. of about 1.1-5, 1.5-3.5 or 2-4, whereas in corresponding non manipulated cells the ratio is lower than 1. In addition, as disclosed herein, PD-1 knockout by conventional (isoform non-specific) gene editing systems directed to PD-1 do not typically induce such an augmentation.

In particular, in resting native primary human T cells, no substantial de-novo transcription of PD-1 occurs in the absence of activation. Upon T cell activation (e.g. TCR mediated), transcription of both flPD-1 and sPD-1 can be initially observed, wherein flPD-1 is preferentially expressed. For example, within three hours of stimulation (TCR mediated e.g. by anti-CD3 antibodies), flPDl transcript levels amounts to about 75-80% of the transcribed PD-1 isoforms, with sPD-1 transcript levels amounting to less than 25-20% (for example in CD4 ⁺ cells). The sPD-1 levels continuously decline thereafter, whereas flPD-1 levels are enhanced within 24 and 48 hrs. of activation. At 72 hrs. of activation, flPD-1 is still transcribed, whereas no substantial sPD-1 transcription is detected. In contradistinction, primary human T cells edited by gene editing systems of the invention are characterized by significant sPD-1 transcript levels (readily detectable by e.g. RT-PCR) within 24, 48 and/or 72 hrs. post TCR mediated activation.

As further disclosed herein, ACT compositions in accordance with the invention exhibit advantageous functional properties as compared to hitherto disclosed ACT compositions. Accordingly, cell compositions of the invention can provide improved therapeutic efficacy and be used in the treatment of new patient populations not hitherto considered amenable for treatment. For example, established ACT compositions based on TIE or other non-engineered autologous or allogeneic lymphocytes, are often reported to have limited efficacy (or to lose efficacy over time) due to e.g. exhaustion mediated by upregulated checkpoint molecules on the adoptively transferred cells. In recent years, ACT protocols employing genetic engineering or gene editing are being developed in an attempt to improve therapeutic efficacy and overcome treatment resistance. However, these newly developed approaches are typically accompanied by challenges with respect to retention over time and long-term efficacy. In the case of the PD-l/PD- L1 axis, additional specific challenges are often reported, in which upon elimination or downregulation of PD-1 or PD-L1 (for example by gene editing-mediated knockout approaches), other checkpoint molecules are up-regulated as a compensatory mechanism, such that the effector cells regain (or retain) their exhausted state. As disclosed herein, the present invention in embodiments thereof relates to advantageous ACT composition that provide an improvement or even substantially overcome hitherto reported challenges associated with ACT.

In some embodiments, the invention relates to non-naturally occurring immune cell populations, and cell compositions comprising same, which at least a portion of the cells is characterized by a splice switching indel mutation associated with exon 3 skipping. In some embodiments, the mutation is associated with NHEJ repair of a double- stranded DNA break between positions 6538-6544, 6539-6540 or 6543-6544 of human PDCD1, wherein each possibility represents a separate embodiment of the invention. In some embodiments, said mutation results in differential modulation of PD-1 isoforms, such that the relative level of the non-membrane bound PD-1 isoform transcript compared to the full-length PD-1 isoform transcript is augmented. In another embodiment said cells are characterized by a transcript ratio of non-membrane bound PD-1 to full-length PD1 that is greater than 1.

Additional embodiments

In one aspect, there is provided a PD-1 -specific expression-modulating nucleic acid molecule, comprising 15-30 contiguous nucleotides that are specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 1. In one embodiment, the molecule (or sequence) is at least 90%, at least 95% or at least 98% complementary to the nucleic acid target, wherein each possibility represents a separate embodiment of the invention.

In another embodiment, the nucleic acid molecule is specifically hybridizable with a nucleic acid target as set forth in SEQ ID NO: 2. In another embodiment, the nucleic acid molecule has the nucleic acid sequence as set forth in SEQ ID NO: 3. In another embodiment, said nucleic acid molecule is an oligonucleotide having the nucleic acid sequence as set forth in SEQ ID NO: 3. In another embodiment, said nucleic acid molecule is a synthetic, non-naturally occurring polynucleotide, comprising the nucleic acid sequence as set forth in SEQ ID NO: 3, linked (at the 5' and/or 3' end) to a heterologous sequence. For example, the nucleic acid molecule may comprise the nucleic acid sequence of SEQ ID NO: 3 as a target homologous sequence (crRNA), linked at its 3' end to a Cas9 nuclease binding (tracrRNA) sequence, as described in further detail and exemplified below.

In another embodiment, the nucleic acid molecule is a single- stranded RNA molecule. In another embodiment, the nucleic acid molecule is derivatized by one or more backbone and/or sugar chemical modifications. In another embodiment, the nucleic acid molecule is an oligonucleotide of 18-22 nucleotides in length. In another embodiment, the nucleic acid molecule is derivatized by one or more backbone and/or sugar chemical modifications. In another embodiment, the oligonucleotide comprises one or more 2' sugar modifications. In various embodiments, the modifications are selected from the group consisting of 2’-O-Methyl (2'-O- Me), 2’-O-methoxyethyl (2'-MOE), and combinations thereof. In another embodiment said oligonucleotide is fully derivatized by 2'-O-Me or 2'-MOE.

According to advantageous embodiments as disclosed herein, the nucleic acid molecule is a splice- switching oligonucleotide. In yet another advantageous embodiment as disclosed herein, the nucleic acid molecule is amenable for use in gene editing, e.g. a synthetic guide RNA (gRNA) molecule. In a particular embodiment, said gRNA comprises a targeting sequence (crRNA) as set forth in SEQ ID NO: 3. In another particular embodiment, the gRNA is a single guide RNA (sgRNA). In an exemplary embodiment, said sgRNA has the nucleic acid sequence as set forth in SEQ ID NO: 4. In another embodiment, there is provided a nucleic acid construct encoding the nucleic acid molecule of the invention. In another embodiment, the construct is an expression vector capable of expressing said nucleic acid molecule in human T cells.

In another aspect, there is provided an engineered, non-naturally occurring CRISPR geneediting system, comprising, or encoding: (i) a gRNA of the invention and (ii) an RNA-guided DNA endonuclease enzyme. In another embodiment, the gene-editing system comprises a first nucleic acid sequence encoding the gRNA, and a second nucleic acid sequence encoding the RNA-guided DNA endonuclease enzyme. In other embodiments the endonuclease is Cas9 or Cpfl, wherein each possibility represents a separate embodiment of the invention. In another embodiment, there is provided a vector comprising the engineered non-naturally occurring geneediting system of the invention. In another embodiment, said vector is a viral vector.

In various embodiments, the invention relates to a nucleic acid molecule, construct, system or vector as disclosed herein, which modulates the expression of at least one PD-1 isoform in T- cells and enhances anti-tumor immunity of said cells. In various embodiments, the invention relates to a nucleic acid molecule, construct, system or vector as disclosed herein, which downregulates the expression of the full-length PD-1 isoform and up-regulates the expression of a non-membrane bound PD-1 isoform. In another embodiment there is provided a host cell comprising the construct, system or vector as described herein, wherein each possibility represents a separate embodiment of the invention.

In another embodiment, there is provided a pharmaceutical composition comprising the nucleic acid molecule, construct, system, vector or host cell as disclosed herein, and optionally a pharmaceutically acceptable carrier, excipient or diluent. Each possibility represents a separate embodiment of the invention. In another embodiment, the invention relates to the pharmaceutical composition for use in the treatment of cancer in a subject in need thereof. For example, without limitation, the subject may be afflicted with e.g. melanoma, renal cell carcinoma, lung cancer, breast cancer, head and neck cancer, bladder cancer, urothelial cancer, pancreatic cancer and cervical cancer. Each possibility represents a separate embodiment of the invention. In another embodiment, the subject is afflicted by a tumor characterized by PD-L1 over-expression. In another embodiment, the subject is further treated by cancer immunotherapy. In another embodiment, the cancer immunotherapy is T-cell mediated immunotherapy. In another embodiment, said pharmaceutical composition is for use in inducing or enhancing anti-tumor immunity in a subject in need thereof. In a particular embodiment, the tumor is a solid tumor.

In another aspect, the invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject, or expressing in cells of said subject, one or more PD-l-specific expression-modulating nucleic acid molecules of the invention. In another aspect, the invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject, or expressing in cells of said subject, a system of the invention.

In some embodiments, the subject is afflicted by a tumor selected from the group consisting of melanoma, renal cell carcinoma, lung cancer, breast cancer, head and neck cancer, bladder cancer, urothelial cancer, pancreatic cancer and cervical cancer. In another embodiment, the subject is afflicted by a tumor characterized by PD-L1 over-expression. In another embodiment, the method further comprises administering to the subject cancer immunotherapy. In another embodiment the cancer immunotherapy is T-cell mediated immunotherapy.

In another aspect, there is provided a method of inducing or enhancing anti-tumor immunity in a subject in need thereof, comprising administering to the subject, or expressing in cells of the subject, one or more PD-l-specific expression-modulating nucleic acid molecules as set forth herein, or a system of the invention. Each possibility represents a separate embodiment of the invention. In some embodiments, the tumor is selected from the group consisting of melanoma, renal cell carcinoma, lung cancer, breast cancer, head and neck cancer, bladder cancer, urothelial cancer, pancreatic cancer and cervical cancer. In another embodiment, the tumor is characterized by PD-L1 over-expression. In another embodiment, the method further comprises administering to the subject cancer immunotherapy. In another embodiment the cancer immunotherapy is T-cell mediated immunotherapy.

In another aspect, there is provided a method of inducing or enhancing splice switching in PD-1 expressing cells, comprising introducing into the cells (administering to, or expressing in said cells), one or more PD-l-specific expression-modulating nucleic acid molecules as set forth herein, or a system of the invention. In another embodiment said cells are T cells. In another embodiment said method is performed in vitro. In another embodiment said method is performed ex vivo. In another embodiment said method is performed in vivo.

In another aspect, there is provided a method for preparing a cell composition for immunotherapy, comprising the step of introducing into a leukocyte population, a PD-l-specific expression-modulating nucleic acid molecule or a system of the invention, in an amount and under conditions suitable for inducing or enhancing splice switching in the leukocyte population.

In various embodiments, the PD-1 expressing cells (or leukocyte population) are selected from the group consisting of peripheral blood mononuclear cells, T cells, B cells, Natural killer cells, antigen presenting cells, dendritic cells and macrophages. For example, without limitation, the cells may be e.g. PBMC, tumor infiltrating leukocytes (TIL), tumor- specific T cell clones, genetically modified T cells, B cells, NK cells, antigen presenting cells and tumor-associated macrophages, macrophages or dendritic cells. Each possibility represents a separate embodiment of the invention.

In another aspect, there is provided a method for preparing a T cell composition for adoptive transfer immunotherapy, comprising the step of introducing into (administering to, or expressing in), a T cell population, a PD-l-specific expression-modulating nucleic acid molecules as set forth herein, or a system of the invention, in an amount and under conditions suitable for inducing or enhancing splice switching in the T cell population.

In another embodiment the method comprises: a. providing a T cell population, b. administering to, or expressing in the T cell population, the PD-l-specific expressionmodulating nucleic acid molecule or system, in an amount and under conditions suitable for inducing or enhancing splice switching in said T cell population, and c. expanding said T cell population, so as to obtain a T cell composition adapted for adoptive transfer immunotherapy comprising an effective amount of the resulting T cell population.

According to exemplary embodiments, said T cell containing population is selected from the group consisting of tumor infiltrating leukocytes (TIL), tumor- specific T cell clones, and genetically modified T cells, wherein each possibility represents a separate embodiment of the invention. In another embodiment, said T cell containing population expresses a CAR or an engineered TCR. In yet another embodiment, said T cell containing population does not express a CAR. In another embodiment said cells do not express an engineered TCR. In another embodiment said cells do not express a CAR or an engineered TCR. In another embodiment, step c is performed prior to step b.

In another embodiment there is provided a cell composition for immunotherapy, prepared by the method. In another embodiment, there is provided a T cell composition for adoptive transfer immunotherapy, prepared by the method. In another embodiment, the composition (e.g. T cell composition) is for use in the treatment of cancer in a subject in need thereof. In another embodiment the subject is afflicted by a tumor selected from the group consisting of melanoma, renal cell carcinoma, lung cancer, breast cancer, head and neck cancer, bladder cancer, urothelial cancer, pancreatic cancer and cervical cancer. In another embodiment the subject is afflicted by a tumor characterized by PD-L1 over-expression. In another embodiment the composition (e.g. T cell composition) is for use in inducing or enhancing anti-tumor immunity in a subject in need thereof. In another embodiment the tumor is a solid tumor.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

Example 1. Generation of nucleic acid constructs modulating PD-1 isoforms

Single guide RNAs (sgRNAs) were designed manually using the sequences of the intronexonjunctions of the immune receptor PD-1. Nearby protospacer adjacent motif (PAM) sequences were located, and the potential gRNAs were designed such that the projected location of the mutation (around the 3' end of the target-binding portion of the guide) is at the intronic junction. Potential sgRNA sequences inducing a mutation farther than 6 bp away from the spliceosome recognition site (located around 2-6 bp from the junction) were eliminated. The sequences of the potential sgRNAs were further evaluated for their uniqueness and determined to have a minimal potential for off-target effects using a commercial guide design tool (Synthego® verify guide design tool).

A sgRNA targeting the intron-exon junction of exon 3 of the PD-1 receptor was identified as a possible agent for creating splicing manipulation of PD-1. The sequence of the guide targetbinding portion (targeting sequence) that was used for cloning into the lentiviral transfer vectors was CTTCTGCCCTTCTCTCTGGA, and its corresponding RNA sequence was CUUCUGCCCUUCUCUCUGGA (SEQ ID NO: 3). A nucleic acid molecule encoding the targeting sequence (SEQ ID NO: 3) was cloned into the lentiviral transfer vectors lentiGuide-Puro (Addgene plasmid #52963) and CRISPseq-BFP-backbone (Addgene plasmid #85707), for transfection to Jurkat cells and PBMC, respectively. Cloning was performed using standard protocol (Ran, F Ann et al. “Genome engineering using the CRISPR-Cas9 system.” Nature protocols vol. 8,11 (2013): 2281-2308. doi:10.1038/nprot.2013.143). The complete sequence of the resulting sgRNA, further comprising the endonuclease-binding elements is as follows: CUUCUGCCCUUCUCUCUGGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC CGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 4).

For the generation of viral particles for transduction, Hek293 cells were grown in a 6-well plate to 80% confluence in 3 ml Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and antibiotics (100 units/ml penicillin, 100 pg/ml streptomycin and 2mM L-glutamine, Gibco) at 37°C in humidified atmosphere and 5% CO2. Each plate was transfected using 1.25 pg of the transfer plasmid, 0.44 pg pMD2.G (Addgene plasmid #12259), 0.81 pg psPAX2 (Addgene plasmid #12260), 7.5 pl of lipofectamine 2000 transfection reagent (ThermoFisher Scientific) and Opti-MEM medium (ThermoFisher Scientific) in a total volume of 250 pl. After 48 hours, supernatants containing the viral particles were harvested and filtered through 0.45 pm syringe filters (Minisart®, Sartorius).

The resulting suspension of viral particles was used for transduction into PD-1 -expressing cells, and found to be capable to modulate PD-1 isoforms differentially in peripheral blood mononuclear cells (PBMC) and T cell lines, as described in Examples 2 and 3 below, respectively. Additional functional evaluation of the above-described gene editing system is provided in Example 5.

Example 2. Differential modulation of PD-1 isoforms in PBMC

Peripheral blood mononuclear cells (PBMC) were purified from buffy coats of healthy donors (Hadassah Medical Center Blood Bank, Israel). Upon experiment, PBMC were thawed and cultured in T-cell media including 300 units/ml IL-2 (Clinigen Healthcare Ltd) and 50 ng/ml aCD3 antibody (clone OKT3, ThermoFisher Scientific) for two days. After the activation, cells were collected and plated with a suspension containing the viral particles generated in Example 1 using the CRISPseq-BFP -backbone vector (5 million cells/well with 10 ml of the viral particles suspension and lOug/ml polybrene) and span down for 1 hour, at 900 RPM. One day post transduction, 5-10 million PBMC were electroporated in a Lonza nucleofection cuvette (VPA-1002) with 50 pg of Cas9 protein (Takara), using the T-007 program.

Control cells were PBMC transduced with a control sgRNA designed such that it does not target any known site in the human genome (Src/ scrambled, non-targeting guide). The targeting sequence that was used for cloning into the lentiviral transfer vectors was GTATTACTGATATTGGTGGG (SEQ ID NO: 19), and the resulting control sgRNA including the endonuclease-binding elements has the following sequence: GUAUUACUGAUAUUGGUGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC CGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 20).

The control cells were transduced and electroporated as described above. BFP+ cells represent cells that were successfully transduced with the sPD-1 sgRNA BFP vector (and therefore express BFP). BFP- cells are the cells that did not express the guide (non-fluorescent, BFP negative cells). Two weeks post transduction and electroporation, surface expression of endogenous membrane-bound PD-1 on intact PBMC were evaluated by flow cytometry, using anti-human PD- 1 antibody (APC conjugated, clone EH12.2H7, Biolegend). In addition, the presence of soluble forms of PD-1 in conditioned media of the cells was assayed by ELISA (DuoSet, R&D systems). The results are presented in Figs. 1A-1B.

As can be seen in Fig. 1A, the number of cells expressing PD-1 on their surface following transduction with the vector encoding the sgRNA comprising SEQ ID NO: 3 ("BFP+"), were significantly reduced compared to the non-transduced cells ("BFP-"), following Cas9 administration. In particular, a 1.6-fold reduction (reduction by 39.64%) in the number of cells expressing full-length PD-1 (flPD-1) on their surface was determined in the cell populations transduced with the specific sgRNA compared to the control cells.

As can be seen in Fig. IB, the reduction in surface PD-1 in the presence of the PD-1- specific gene editing system (including the encoded sgRNA comprising SEQ ID NO: 3, "sgRNAl") was concomitant to an enhancement in soluble form(s) of PD-1 (sPD-1) secreted by the cells to the culture media, compared to cells transduced with the non-targeting gene editing system ("Ctrl"). In particular, the levels of sPD-1 secreted by the manipulated cells was about 1.32- fold higher (enhanced by 32.4%) as comparted to the control cells.

The results demonstrate that the PD-l-specific CRISPR/Cas9 editing system was able to manipulate the expression of PD-1 isoforms in PBMC differentially. Without wishing to be bound by a specific theory or mechanism of action, a double-strand break was introduced into a predefined splicing recognition sequence of the PD-1 gene (PDCDF), thereby inducing or enhancing exon 3 skipping. Accordingly, in the manipulated cells, the level of full-length, surface-expressed PD-1 (flPD-1) was reduced, while the levels of soluble PD-1 isoforms were remarkably enhanced.

As disclosed herein, and without wishing to be bound by a specific theory or mechanism, the splice- shifting resulted in the generation of a soluble form of PD-1, in which exon 3 (encoding the transmembrane domain) of the gene is excluded (skipped). As a result of the missing transmembrane domain, the protein may be secreted from the cell.

Example 3. T cells manipulated to express higher levels of sPD-1 exhibit improved functionality

The splicing manipulation of PD-1 was also tested in a T cell line, namely in Jurkat CD4 ⁺ T cells. Jurkat cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco) and antibiotics (100 units/ml penicillin, 100 pg/ml streptomycin and 2mM L-glutamine, Gibco) at 37°C in a humidified atmosphere and 5% CO2. The cells were transduced with the suspension of viral particles comprising a transfer vector expressing the Cas9 protein and a blasticidin resistance gene (Addgene # 52962), using the protocol essentially as described in Example 1. Jurkat cells were grown for 1 week with Blasticidin (Sigma) in order to select for resistant cells, and then transduced a second time with the lentiGuide-Puro transfer plasmid encoding the sgRNA comprising SEQ ID NO: 3, as described in Example 1. The cells were then selected again by incubation in the presence of Puromycin (Sigma) for one week.

Next, the levels of the mRNA transcripts of the PD-1 isoforms were evaluated by qPCR. To this end, cells were activated for 48 hours by adding 200ng/ml Phorbol 12-myristate 13-acetate (PMA, Sigma) and 300ng/ml lonomycin (Sigma) to the media. Then, RNA was isolated from the cells using the commercial kit GenElute mammalian total RNA miniprep (Sigma Aldrich) and reverse-transcription (RT) was performed using the commercial kit qScript cDNA Synthesis Kit (QuantaBio). qRT-PCRs were performed on a Viia7 Real-Time PCR System (Life technologies) using PowerUp SYBR Green Master Mix reagent (Thermo Fischer). Each sample was run in triplicate and normalized to a housekeeping gene, P-actin. Changes in gene expression were calculated by the AACT-method using mean cycle threshold values and the housekeeping gene P-actin. One of the primers designed for detecting flPD-1 is located inside exon 3, which is missing in sPD-1 (Forward - AGATCAAAGAGAGCCTGCGG, Reverse - ACCACCAGGGTTTGGAACTG, SEQ ID NOs: 5 and 6, respectively). One of the primers designed to detect sPD-1 is located on the junction between exon 2 and 4, and therefore can only detect mRNA of isoforms missing exon 3 (Forward - AGGGTGACAGGGACAATAGGA, Reverse - TAGTCCACAGAGAACACAGGC, SEQ ID NOs: 7 and 8 respectively). The results are presented in Fig. 2 A as fold changes relative to the control group (corresponding to cells transduced with a nontargeting guide RNA (described in Example 2, "Ctrl").

As can be seen in Fig. 2 A, the levels of the full-length PD-1 isoform (flPD-1) transcript as measured by qPCR were decreased in cells treated by the PD-1 -specific gene editing system ("sgRNAl") compared to cells treated by non-targeting guide ("Ctrl"). In parallel, a remarkable, highly significant enhancement in the transcript levels of truncated PD-1 (sPD-1) was recorded. In particular, a twofold reduction in the flPD-1 transcript and a threefold enhancement in the sPD- 1 transcript levels were measured. These results demonstrate that the sgRNA transduction led to increased splicing manipulation, exon 3 skipping and modulation of PD-1 isoforms at the mRNA transcript level.

Fig. 2B shows the results of a flow cytometry analysis performed essentially as described in Example 2, in which an additional control of PD-1 knockout Jurkat cells (PD-1 KO, black line) was used. These PD-1 KO control cells were prepared essentially as described in Example 2, with a sgRNA designed to downregulate the expression of all PD-1 isoforms, having a targeting sequence as follows: CGACTGGCCAGGGCGCCTGT (SEQ ID NO: 9, or CGACUGGCCAGGGCGCCUGU in the resulting sgRNA molecule, wherein the entire sgRNA molecule of the PD-1 KO control system has the sequence as set forth in SEQ ID NO: 10, as follows: CGACUGGCCAGGGCGCCUGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC CGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC). As can be seen, while treatment with the PD-1 isoform-specific gene editing system (sgRNAl, dashed line) reduced the level of flPD-1 as compared to the non-manipulated cells, the levels of flPD-1 were still significantly higher than those measured in PD-1 KO cells. Scramble sgRNA was used as a control ("Ctrl", horizontal stripes) and the unstained cells as the background ("unstained", vertical lines).

Changes in mRNA transcript levels of flPD-1 and sPD-1 in Jurkat cells treated by the PD- 1 isoform-specific gene editing system (sgRNAl) compared to cells treated by non-targeting guide (control) and the PD-1 KO guide as described above, were also evaluated by agarose gel electrophoresis of PCR product, and band intensities were quantified using the ImageJ analysis tool. It was found that the transcript ratio of sPD-1 to flPDl in the control and PD-1 KO groups were both below 1 - a ratio of 0.65 was measured in the control group, which was further reduced to 0.27 in the PD-1 KO group. In contradistinction, the gene editing system comprising sgRNAl induced a remarkable enhancement in the sPD-1 to flPDl ratio, which was increased to 2.69. Thus, differential modulation of PD-1 isoform expression by the splice- switching gene editing system markedly augmented the sPD-1 to flPDl ratio, whereas no such augmentation was observed upon PD-1 knockout by a conventional PD-1 (isoform non-specific) gene editing system (which rather even decreased this ratio further).

Next, the changes in PD-1 expression levels were evaluated in another Jurkat cell line, engineered to express a melanoma- specific targeting TCR (directed to the NY-ESO-1 tumor- associated antigen) and the Cas9 protein and a blasticidin resistance gene (Addgene # 52962), using the protocol essentially as described in Example 1. Cells treated by the PD-1 -specific gene editing system ("sgRNAl", dashed line), cells treated by non-targeting guide ("Ctrl", horizontal stripes) and PD-1 knockout cells (PD-1 KO, black line) were co-cultured for 48 hrs. with T2 cells loaded with Ipg/ml of NY-ESO1-1 peptide. Unstained cell used as the background ("unstained", vertical stripes) Fig. 2C shows the results of a flow cytometry analysis performed for PD-1 surface expression on the Jurkat cells. As can be seen, while treatment with the PD-1 isoform-specific gene editing system reduced the level of flPD-1 as compared to the non-manipulated cells, the levels of flPD-1 were still significantly higher than those measured in PD-1 KO cells.

Next, the functionality of the cells of the different treatment groups was evaluated by examining their ability to secrete IL-2 in response to activation stimuli. To this end, cells were activated for 48 hours by adding 200ng/ml Phorbol 12-myristate 13-acetate (PMA, Sigma) and 300ng/ml lonomycin (Sigma) to the media, and IL-2 secretion was assayed by ELISA (DuoSet, R&D systems). As can be seen in Fig. 2D, IL-2 secretion was remarkably enhanced in cells treated by the PD-1 isoform-specific gene editing system ("sgRNAl") compared to the other treatment groups. In particular, the sgRNAl group exhibited a 1.88-fold enhancement in secreted IL-2 levels (enhancement by 89.25%) compared to the control group treated with non-targeting gene editing system. In contradistinction, in the PD-1 KO group, no such enhancement in IL-2 secretion as compared to the control was measured, and the PD-1 KO cells even appeared to secrete slightly lower levels of IL-2 than the control group.

Accordingly, the results in Figs. 2A-2D show a correlation between the level of activation- induced IL-2 secretion and the level of de-novo sPD-1 transcription, and not to the level of flPD- 1 transcription. Thus, the results demonstrate that the unexpectedly improved functionality of the cells associated with the differential modulation of PD-1 isoforms was due to the elevated sPD-1 levels and not to the loss of the PD-1 receptor (flPD-1).

Normally, T-cells expressing flPD-1 may be inhibited by PD-L1, commonly expressed on cancer cells (schematically illustrated in Fig. 3A). As shown in the above examples, and without wishing to be bound by a specific theory or mechanism of action, alternative splicing manipulation of PD-1 according to the present invention may induce or enhance secretion of soluble forms of PD-1 from T cells, resulting in PD-L1 blockage and thereby improved T-cell functionality and enhanced anti-tumor immune response (schematically illustrated in Fig. 3B).

Example 4. PD-l-specific splice-switching oligonucleotides

Based on the in-silico and in-vitro analyses, the target sequence for expression-modulating antisense oligonucleotides (ASOs) within the PD-1 transcript was identified as having the nucleic acid sequence as set forth in SEQ ID NO: 1, as follows: TTTGTGCCCTTCCAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGC (SEQ ID NO: 1). ASOs 15-30-mer-long, designed to target (specifically hybridize with) partially overlapping regions of SEQ ID NO: 1, are synthesized using 2-0-Me modified nucleosides.

For transfection into Jurkat T cells, 5pM of the ASO are transfected into 5xl0 ⁶ cells in 330pl RPMI medium, by electroporation in 0.2cm cuvettes (Biorad) using ECM 630 Electro Cell manipulator (BTX Harvard apparatus) Exponential electroporation - 250V, 300pF, 1000Q. Twenty-four hours later, RNA is extracted using a commercial RNA extraction kit such as GenElute Mammalian Total RNA kit (Sigma, RTN70) according to the manufacturer’s protocol. PD-1 isoforms are then detected by flow cytometry and/or qPCR, and their functionality is evaluated by measuring activation-induced IL-2 secretion, as described in Example 3 above.

For transfection into PBMC, 5pM of the ASO are transfected to PBMC from healthy donors (5xl0 ⁶ cells in lOOul of commercial electroporation buffer) by electroporation in 0.2cm cuvettes (Lonza) using AMAXA nucleofector 2b (LONZA). PD-1 isoforms are then detected by flow cytometry and/or qPCR, and their functionality is evaluated by measuring activation-induced cytokines (IFNy) secretion.

Example 5. Improved anti-tumor activity in tumor-specific lymphocytes following splicing manipulation

In order to evaluate the functionality of tumor- specific effector cells following splicing manipulation by the PD-1 isoform-specific gene editing system, a model of PBMC transduced with a melanoma- specific TCR was used. To this end, PBMC purified from huffy coats of healthy donors were thawed and cultured in T-cell media including: 300 units/ml IL-2, 5 g/ml platebound aCD3 antibody (clone OKT3, ThermoFisher Scientific) and 5 pg/ml soluble aCD28 antibody (clone CD28.2) for two days. After the activation, cells were collected and plated on an untreated tissue culture plate (SPL, life science) coated with retro-viral particles encoding for a melanoma-specific targeting TCR (directed to the NY-ESO-1 tumor-associated antigen). The plated PBMC were supplemented with a suspension containing the viral particles, generated as detailed in Example 1 (i.e., lentiGuide-Puro, Plasmid #52963 - Addgene), 2.5 million cells/well with 5 ml of the suspension of viral particles and lOpg/ml polybrene, and span down for 1 hour, at 900 RPM. One-day post transduction, 5-10 million PBMC were electroporated in a Lonza nucleofection cuvette (VPA-1002) with 10pg/ 10 ⁶ cells of Cas9 protein (Takara), using the T-007 program.

Accordingly, the test group ("PD-1 sgRNAl") included cells transduced with both the melanoma-specific TCR and the gene editing system comprising the PD-1 isoform-specific sgRNA (sgRNAl, comprising SEQ ID NO: 3). The control cells included two groups of PBMC transduced and electroporated as described above, with the exception that the transduced gRNAs were either a non-targeting guide as described in Example 2 above ("SCR control") or the sgRNA comprising SEQ ID NO: 9 as described in Example 3 above ("PD-1 KO").

Four days post transduction and electroporation, cells were re-activated with 300 units/ml IL-2, 0.1 pg/ml plate-bound aCD3 antibody and 0.1 pg/ml soluble aCD28 antibody for 24 hours. The following day the cells were co-cultured with either the wild-type (WT) melanoma cell line Mel 624, or with Mel 624 cells engineered to overexpress PD-L1 ("Mel 624-PD-L1"). The cytotoxic capacity of the cells was evaluated by intracellular staining of cleaved caspase3 in the target melanoma cells as an indicator of early apoptosis. To this end, the Mel 624 and Mel 624- PD-L1 cells were pre-stained with the CellTrace Far Red DDAO-SE dye (Life Technologies, allowing easy detection of the melanoma cell population in flow-cytometry) according to the manufacturer's protocol. Labeled melanoma cells were co-cultured with effector lymphocytes for 90 minutes at a 1:1 effector : target ratio. The cells were then washed, fixed, permeabilized (BD protocol), and labeled with rabbit anti-cleaved caspase-3-PE (BD Pharmingen). Washed cells were subjected to flow cytometry. The results are presented in Fig. 4.

As can be seen in Fig 4, PD-1 expression modulation associated with either differential modulation of PD-1 isoforms (sgRNAl) or with complete PD-1 knockout (KO) resulted in enhanced cytotoxicity and anti-tumor activity. The response was not obligatory to tumor cells overexpressing PD-L1, as it was also observed when the lymphocytes were incubated with WT Mel 624 target cells, expressing endogenous low levels of PD-L1. In particular, enhancement of about twofold against low-PDL-1 expressing tumor cells and of about 1.3-fold against PDL-1 overexpressing tumor cells, were observed in tumor- specific PBMC treated with a gene editing system having a sgRNA comprising SEQ ID NO: 3 as compared to those treated with a system comprising a non-targeting control.

Thus, despite expressing significantly higher levels of full-length PD-1 than the PD-1 KO cells (Fig. 2B), PBMC treated with the splicing-modulating gene editing system were at least as effective at enhancing anti-tumor immunity and cytotoxicity as PBMC substantially lacking PD-1 expression.

Example 6. Differential modulation of PD-1 isoforms utilizing gene editing Ribonucleic Proteins (RNPs)

For the preparation of PD-1 specific gene editing RNPs, a gRNA having a targeting sequence as detailed in Example 1 and the Cas9 endonuclease protein were combined to provide a ribonucleic protein (RNP). Briefly, a PD-1 specific crRNA comprising SEQ ID NO: 3 followed by a 16-nucleotide long (16-nt) annealing sequence at its 3' end, and an annealable universal 67- nt Cas-binding tracrRNA, were ordered from IDT (Integrated DNA Technologies Inc.). The crRNA and tracrRNA were resuspended in IDT buffer and mixed at equimolar concentrations to a final concentration of lOOpM. The resulting crRNA-tracrRNA complex (gRNA) was then combined with Cas9 protein (104 pmol of Cas9 with 120 pmol of crRNA-tracrRNA complex) in 25p I total volume, to form PD-1 isoform-specific RNPs.

The ability of the resulting RNPs to modulate the expression of PD-1 isoforms was tested in primary tumor-infiltrating lymphocytes (TIL) extracted from melanoma patient (herein designated TIL 209 cells). The TIL were thawed and cultured for 24 hours in T-cell media containing 3000 units/ml IL-2. After 24 hours, 5-10 million cells were electroporated in a Lonza nucleofection cuvette (VPA-1002) with the RNPs, using the U-014 program. As controls, RNPs comprising the PD-1 KO crRNA and non-targeting crRNA (SCR) as described in Examples 2 and 3 (SEQ ID NOs: 9 and 19, respectively) were used.

24 hours post electroporation, the TIL were activated with 0.1 pg / ml plate-bound anti- CD3 antibody (OKT3) and O.lpg / ml soluble anti-CD28 for 24 hours, and then co-cultured with the two 624 melanoma lines, Mel 624 and Mel 624-PD-L1. Incubation was performed for 1.5 hours for the cleaved caspase 3 killing assay, and for 24 hours for evaluating the levels of PD-1 isoforms. The results are presented in Figs. 5A (fl PD-1 levels), 5B (sPD-1 secretion), and 5C (cytotoxic activity). Optionally, an anti PDL-1 blocking antibody (Atezolizumab, Tecentriq, Roche, 20 pg/ml) was added to some of the wells as indicated in Fig. 5B ("atezo").

Fig. 5A presents the expression levels of flPD-1 measured by flow cytometry. TIL introduced with either the PD-1 isoform-specific gene editing RNPs (“RNP gRNAl”), the scrambled non-targeting guide (“RNP Scr”) or the PD-1 KO guide (“RNP PD-1 KO”). As can be seen, the RNP gRNAl group demonstrated lower flPD-1 levels compared to the non-targeting control group.

Fig. 5B presents the expression levels of sPD-1 measured by ELISA, showing increased levels of sPD-1 in the splice manipulated cells with an additional elevation when anti PD-L1 antibody was added to the cells (“Atezo”). In particular, a 1.34-fold enhancement (34.09%) in sPD-1 levels over the control is shown in the RNP gRNAl group, wherein in the presence of the anti-PD-Ll antibody an enhancement of 1.51-fold (51.11%) is shown. In contradistinction, sPD-1 levels were decreased in the RNP PD-1 KO group compared to the control (RNP Scr) group. In addition, the results indicate that the sPD-1 isoform produced following the splice manipulation retain its functionality in binding its ligand PD-L1, as can be determined from the elevated levels of sPD-1 in the culture medium measured in the presence of the anti-PD-Ll antibody.

Fig. 5C presents the cytotoxic capacity of the TIL evaluated by FACS, calculated as percentile of cleaved caspase-3 expression as an indicator of early apoptosis, essentially as described in Example 5. As can be seen, The PD-1 splicing manipulation in T cells was also associated with improved cytotoxic capacity, manifested as a higher percentage of melanoma cells labeled with cleaved caspase 3. In particular, a 1.7-fold (71.73%) enhancement in the cytotoxic capacity against low-PD-Ll expressing cells and a 3.85-fold (285.71%) enhancement in the cytotoxic capacity against PD-L1 overexpressing cells was observed (compared to control TIL treated by non-targeting RNPs).

As disclosed herein, and without wishing to be bound by a specific theory or mechanism of action, the introduction of the RNP-based PD-1 isoform-specific gene editing system promoted splice- switching in human primary TIL, resulting in a decrease of the flPD-1 isoform and a remarkable increase of a sPD-1 isoform, in which exon 3 (encoding the transmembrane domain) of the gene is excluded (skipped). This was accompanied by enhanced anti-tumor immunity, in particular manifested as enhanced cytotoxic capacity, not only against tumor cells overexpressing PD-L1 but also of tumor cells that express low endogenous PD-L1 levels. Example 7. Development and characterization of additional advantageous gene editing systems for modulating splice isoforms

Additional gene editing systems comprising different gRNA sequences were synthesized as RNPs essentially as described in Example 5. The sequences of the targeting sequence of the additional tested gRNAs (crRNAs) were as follows: UUGUGCCCUUCCAGAGAGAA (gRNA2, SEQ ID NO: 11); UUUGUGCCCUUCCAGAGAGA (gRNA3, SEQ ID NO: 13);

UUCUGCCCUUCUCUCUGGAA (gRNA4, SEQ ID NO: 15); and GGCACUUCUGCCCUUCUCUC (gRNA5, SEQ ID NO: 17).

RNPs incorporating these sequences were analyzed for PD-1 expression and tumorspecific activity essentially as described in Example 6, and compared to RNPs comprising gRNAl (targeting sequence comprising SEQ ID NO: 3, Example 6). To this end, TIL 209 cells were thawed and cultured for 24 hours in T-cell media containing 3000 units/ml IL-2. After 24 hours, 5-10 million cells were electroporated in a Lonza nucleofection cuvette (VPA-1002) with RNPs, using the U-014 program. The scrambled guide described in Example 5 ("scr") was used as a control. 24 hours post electroporation, TILs were activated with Ip/ml plate-bound aCD3 antibody (OKT3) and Ipg/ml aCD28 antibody for an additional 24 hours followed by flow cytometry analysis to evaluate flPD-1 expression level. The results identified gRNA4 and gRNA5 as additional gRNAs having advantageous properties (as shown for previously characterized gRNAl), compared to the other tested gRNAs. Representative results for the particularly advantageous gRNAs are presented in Figs. 6A-6D, as detailed below.

In particular, Figs. 6A-6C show FACS histograms of the expression levels of flPD-1 following administrations of RNPs comprising gRNAs of SEQ ID NOs: 3, 15, and 17, respectively. Quantitative representation of these results is provided in Fig. 6D, which summarizes mean fluoresce intensity (MFI) of the histograms presented in Figs 6A-6C. As can be seen, gRNAs of SEQ ID NOs: 15 and 17 were at least as potent as the gRNA of SEQ ID NO: 3 in downregulating flPDl expression (downregulation by 1.63-, 1.31- and 1.32-fold, respectively).

It is noted, that the additional gRNA sequences identified as being particularly advantageous, namely SEQ ID NOs: 15 and 17, substantially overlap with SEQ ID NO: 3, such that they are complementary to 19 or 16 contiguous nucleotides of the target sequence of SEQ ID NO: 2, respectively. As such, SEQ ID NOs: 3, 15 and 17 contain 20, 19 or 16 contiguous nucleotides, respectively, that are specifically hybridizable with the nucleic acid target of SEQ ID NO: 2. Accordingly, SEQ ID NO: 2 was identified as a preferable target for differential modulation of PD-1 isoforms using gene editing CRISPR systems. Example 8. PD-1 splicing manipulation of human lymphocytes in in vivo models

The splicing manipulation of PD-1 in human lymphocytes is evaluated in vivo using the Winn mouse model. The model utilizes NSG mice, that carry two mutations - severe combined immune deficiency (scid) and a complete null allele of the IL2 receptor common gamma chain (IL2rg ^nul1 '). The scid and IL2rg ^nul1 mutations render the mice B and T cell deficient and prevents cytokine signaling through multiple receptors, leading to a deficiency in functional NK cells. The severe immunodeficiency allows the mice to be humanized by engraftment of human CD34 ⁺ hematopoietic stem cells, PBMC, patient-derived xenografts, or adult stem cells and tissues. To this end, TIL 209 cells are electroporated with RNPs essentially as described in Example 6. Next, 10 ⁶ of the TIL introduced with the RNPs are washed and mixed at a 1:1 ratio with human melanoma cells, and immediately injected subcutaneously into the back of 8- to 10-week-old female NSG mice. Tumor size is measured in two perpendicular diameters three times per week. Mice are sacrificed when tumors reach a 15-mm diameter in one dimension or when the lesion necrotizes.

The effect of the splice- switching RNPs are further evaluated in vivo, using an adoptive cell therapy (ACT) mouse model. The model utilizes NSG mice bearing human melanoma, in which the human melanoma is either the wild-type tumor (expressing low levels of PD-L1) or the genetically modified melanoma cells engineered to over express PD-L1. To this end, human melanoma cells (wild-type or PD-L1 over expressing) are injected subcutaneously into the back of 8- to 10-week-old female NSG mice. In parallel, TIL 209 cells are electroporated with RNPs essentially as described in Example 6 (comprising PD-1 splicing modulating system, PD-1 KO and non-targeting control (SCR). When the tumors are palpable, 5-10xl0 ⁶ of the TIL introduced with the RNPs are washed and injected intravenously. Tumor size is measured in two perpendicular diameters three times per week. Representative mice from the different treatment groups are sacrificed after 1, 2 and 3 weeks after treatment initiation for analysis of the persistence and localization of the adoptively transferred T cells. Additionally, representative mice will be followed until tumors reach a 15-mm diameter in one dimension or when the lesion necrotizes to analyze mice survival.

Thus, the ACT model facilitates determining the effect of the splice- switching gene editing system in an established tumor model, evaluating the long-term effects of the splicing manipulation on the treated lymphocytes in an in vivo model as compared to PD-1 -knockout approach, and comparative evaluation of the effect on the tumor as a function of its PD-L1 expression level. In summary, disclosed herein is the construction of various PD-1 isoform-specific gene editing systems, that are capable of modulating the expression of PD-1 isoforms in a differential manner. In particular, administration of the systems either in the form of expression constructs introduced using a viral vector in combination with a Cas9 endonuclease, or as RNP complexes, were capable of significantly downregulating the expression of flPD-1 while significantly upregulating the expression of sPD-1, at both the transcript and protein levels. This unique modulation pattern is distinct from those associated with previously tested approaches such as conventional gene editing systems aimed at eliminating or downregulating the expression of PD- 1 in a non-differential manner. Further, this expression pattern was associated with a remarkable enhancement in activation-induced cytokine secretion and with improved anti-tumor immunity and cytotoxicity.

It is noted, that hitherto suggested approaches of PD-1 modulation for therapeutic purposes are typically associated with the development of tumor resistance, even if initially effective. This obstacle impairs the use of PD-1 modulators such as immune checkpoint inhibitors and genetic knockout of PDCD1 using gene editing systems in clinical practice. As disclosed herein, advantageous gene editing systems of the invention as characterized hereinabove provide for an improved therapeutic modality, targeting both the (full-length) PD-1 receptor on effector immune cells, and PD-L1 expressed by tumor cells (via sPD-1 secretion). Without wishing to be bound by a specific theory or mechanism of action, advantageous gene editing systems of the invention may thus be readily used in long-term therapeutic regimens, while substantially reducing the risk of resistance, and while not obligatorily depending on the expression of the receptor on immune cells and its ligand on tumor cells for their activity.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.