FIELD OF THE INVENTION

The present invention relates to a non-integrating expression vector for use in a repeated administration regimen for non-intergrating gene therapy, wherein said vector does not induce a long-term adaptive immune response in the patient treated with the gene therapy. The non-integrating expression vector for use in a repeated administration regimen for non-integrating gene therapy comprises a ONA sequence encoding an E2 protein of Bovine Papilloma Virus type 1 (BPV1), comprising a DNA-binding dimerization domain, said E2 protein being operatively linked to a heterologous promoter, as well as an oligomerized DNA sequence, forming a binding site for an E2 protein of Bovine Papilloma Virus type 1 (BPV1).

In some embodiments, at least 50% of all immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter of said non-integrating expression vector are further removed and/or modified in such a way that they do not activate an in- nate immune response in a mammalian cell. The present invention also relates to the new vectors as such as well as to their uses in a repeated administration regimen for non-intergrating gene therapy.

The non-integrating expression vector(s) described herein further comprise one or more expression cassette(s) of a DNA sequence of interest being operatively linked to a heterologous promoter.

In some embodiments, the present invention in particular relates to a non-integrating expression vector, which does not induce a long-term adaptive immune response in a patient, comprising one or more expression cassette(s) encoding a protein or fragments) thereof binding an antigen associated with cancer, tumor(s) and/or solid tu- mor(s) and/or cell signaling associated with said cancer, tumor(s) and/or solid tu- mor(s). The present invention consequently also relates to said non-integrating expression vector for use in expressing a protein or fragment(s) thereof binding an antigen associated with cancer, tumor(s) and/or solid tumor(s) and/or cell signaling associated with said cancer, tumor(s) and/or solid tumor(s) as well as to a gene therapeutic containing said vector, to methods for the preparation of the vector and to therapeutic uses of said vector, in particular for non-intergrating gene therapy for cancer treatment.

A non-integrating expression vector according to the present invention is particularly well suited for gene-therapy treatments wherein an expression of a therapeutic magni- tude and moderate durability is desired, such as for use in cancer treatment, such as in particular for passive immunotherapy for cancer.

BACKGROUND OF THE INVENTION

Gene therapy, i.e. transfer of autologous or heterologous genes into animal or human organisms with suitable vectors is emerging as a technique with immense potential to cure diseases with a genetic background or to prevent and/or cure infectious diseases.

Different gene therapy platforms are tailored to different disease targets. In general, one distinguishes between integrating and non-integrating gene therapy strategies and/or gene transfer. Non-integrating gene transfer has the advantage that the expression of the gene of interest can be directely controlled by administration dose and regimen and also be terminated. Furthermore, there is a lesser risk for oncogenecity. Non-integrating gene delivery vectors that do not rely on host cell genome integration offer several advantages for gene transfer, chiefly the avoidance of insertional mutagenesis and position effect variegation. However, unless engineered for replication and segregation, non-integrating vectors will dilute progressively in proliferating cells, and are not exempt of epigenetic effects. In particular long term and/or life long treatment with non-integrating gene therapies is often hampered by an ever increasing immunological response to the vector elements of non-human and/or trans-species origin. Several types of viral and non-viral vectors have been developed and tested in animals and in human subjects to deliver a gene or several genes that is (are) defective by mutation and therefore non-functional. Examples of such vectors include Adenovirus vectors, Herpes virus vectors, Retrovirus vectors, Lentivirus vectors and Adeno- associated vectors.

Such vectors have in particular been used for vaccine purposes. Since 1990, DNA immunization has become a standard method to induce immune responses to foreign proteins in experimental animal and human studies. Such vectors are called DNA vaccines.

Generally, the DNA vectors used in vaccines contain a cloning site for the gene of interest, a strong viral promoter, such as the immediate early promoter of the CMV virus, in order to drive the expression of the gene of interest, a polyadenylation region, and an antibiotic resistance gene and a bacterial replication origin for the propagation of the DNA vector (plasmid) in bacterial cells.

With the vectors described above it is possible to obtain a detectable level of expression of the gene of interest after administering the vector to experimental animals or to humans, either by a direct injection to muscle or to skin with a particle bombardment technique or by applying the vector in a solution directly to mucous membranes. However, the expression obtained by these vectors is short lived: the vectors tend to disappear from the transfected cells little by little and are not transferred to daughter cells in a dividing cell population. The short-term expression of the gene of interest and lim- ted number of cells targeted are probably the major reasons, why only temporary immune responses are observed in subjects immunized with DNA vaccine vectors described above.

Clearly, many technical challenges must still be overcome before gene therapy can be a practical approach to treating diseases, especially regarding gene therapies that require longterm or even for life-long treatments. Better ways to deliver genes and to target them to particular cells, as well as a better ability to control gene expression in the context of gene transfer is sought after. What is more, immune responses to introduced elements that are not human in origin are an issue particularly troublesome in repeated administration regimens needed for effective long-term non-integrating gene therapy.

There is a growing interest in developing novel products useful in gene therapy and DNA vaccination. For instance, papilloma virus vectors carrying the expression cassette for a gene of interest have been suggested to be useful candidates.

To date more than 70 subtypes of human papilloma viruses (HPVs) and many different animal papilloma viruses have been identified. All papilloma viruses share a simi- lar genome organization and the positioning of all of the translational open reading frames (ORFs) is highly conserved. Papilloma viruses infect squamous epithelial cells of skin or mucosa at different body sites and induce the formation of benign tumors, which in some cases can progress to malignancy. The papilloma virus genomes are replicated and maintained in the infected cells as multicopy nuclear plasmids. The replication, episomal maintenance, expression of the late genes and virus assembly are tightly coupled to the differentiation of the epithelial tissue: the papilloma virus DNA episomal replication takes place during the initial amplificational replication and the second, i.e. latent, and the third, i.e. vegetative, replications in the differentiating epithelium.

Two viral factors encoded by the E1 and E2 open reading frames have been shown to be necessary and sufficient for the initiation of the DNA replication from the papilloma virus origin in the cells (Ustav, M. and Stenlund, A., EMBO J 10 (1991) 449 - 57; Ustav, M., et al., EMBO J 10 (1991) 4321 - 4329; Ustav, E., et al., Proc Natl Acad Sci USA 90 (1993) 898 - 902).

Functional origins for the initiation of the DNA replication have been defined for BPV1 (Ustav, M., et al., EMBO J 10 (1991) 4321 - 4329], HPV1a [Gopalakrishnan, V. and Khan, S., supra], HPV11 [Russell, J., Botchan, M., J Virol 69 (1995) 651 -660], HPV18 [Sverdrup, F. and Khan, S., J Virol 69 (1995) 1319 - 1323:Sverdrup, F. and Khan, S., J Virol 68 (1994) 505-509) and many others. Characteristically, all these origin fragments have a high A/T content, and they contain several overlapping individual E1 protein recognition sequences, which together constitute the E1 binding site (Ustav, M., et al., EMBO J 10 (1991) 4321 - 4329; Holt, S., et al., J Virol 68 (1994) 1094 - 1102; Holt, S. and Wilson, V., J Virol 69 (1995) 6525 - 3652; Sedman, T., ef al. J Virol 71 (1997) 2887 - 2996). In addition, these functional origin fragments contain an E2 binding site, which is essential for the initiation of DMA replication in vivo in most cases (Ustav, E., ef a/., Proc Natl Acad Sci U S A. 1993 Feb 1 ;90(3):898-902). The E2 protein facilitates the first step of the origin recognition by E1. After the initial binding of monomelic E1 to the origin, the multimerization of E1 is initiated. This leads to the formation of the complex with the ori melting activity. It has been suggested that E2 has no influence on the following stages of the initiation of the DNA replication (Lusky, M., et al., Proc Natl Acad Sci USA 91 (1994) 8895-8899). The BPV1 E2 ORF encodes three proteins that originate from selective promoter usage and alternative mRNA splicing (Lambert, P., ef a/., Annu Rev Genet 22 (1988) 235 - 258). All these proteins can form homo- and heterodimers with each other and bind specifically to a 12 bp interrupted palindromic sequence 5'-ACCNNNNNNGGT-3' (SEQ.ID.NO.:12) (Androphy, E., ef a/., Nature 325 (1987) 70 - 739).

There are 17 E2 binding sites in the BPV1 genome and up to four sites in the HPV genomes, which play a crucial role in the initiation of viral DNA replication (Ustav, E., ef al., Proc Natl Acad Sci U S A. 1993 Feb 1 ;90(3):898-902) and in the regulation of viral gene expression (Howley, P. M., Papillomavirinae: the viruses and their replica- tion, in Virology, Fields, B. C, Knipe, D. M., Howley, P. M., Eds., Philadelphia: Lip- pincott-Raven Publishers, 1996. 2. edition, p. 2045 - 2076). Structural and mutational analyses have revealed three distinct functional domains in the full size E2 protein. The N-terminal part (residues 1 to 210) is an activation domain for transcription and replication. It is followed by the unstructured hinge region (residues 211 to 324) and the carboxy-terminal DNA binding-dimerization domain (residues 325 to 410) (Dostat- ni, N., ef a/., EMBO J 7 (1988) 3807 - 3816; Haugen, T., ef al. EMBO J 7 (1988) 4245 - 4253; McBride, A., et al., EMBO J 7 (1988) 533 - 539; McBride, A., ef al., Proc Natl Acad Sci USA 86 (1989) 510-514). On the basis of X-ray crystallographical data, the DNA binding-dimerization domain of E2 has a structure of a dyad-symmetric eight- stranded antiparallel beta barrel, made up of two identical "half-barrel" subunits

(Hegde, R., ef al., Nature 359 (1992) 505 - 512; Hegde, R., J Nucl Med 36(6 Suppl) (1995) 25S - 27S). The functional elements of the transactivation domain of E2 have a very high structural integrity as confirmed by mutational analysis (Abroi, A., ef al., J Virol 70 (1996) 6169 - 6179; Brokaw, J., ef al., J Virol 71 (1996) 23 - 29; Grossel, M., et al., J Virol 70 (1996) 7264 - 7269; Ferguson, M. and Botchan, M., J Virol 70 (1996) 4193-4199) and by X-ray crystallography (Harris, S., and Botchan, M R., Science 284 (1999) 1673-1677 and Antson, A. et al., Nature 403 (2000) 805-809). In addition, X- ray crystallography shows that the N-terminal domain of the ΕΞ2 protein forms a dimer- ic structure, where Arg 37 has an important function in dimer formation (Antson, A., et a/., supra).

As has been described previously, bovine papillomavirus type 1 E2 protein in trans and its multiple binding sites in cis are both necessary and sufficient for the chromatin attachment of the episomal genetic elements. The phenomenon is suggested to pro- vide a mechanism for partitioning viral genome during viral infection in the dividing cells (lives, I., ef a/., J Virol. 73 (1999) 4404-4412).

The present inventors in WO02/090558 for the first time presented that expression vectors, which carry (A) an expression cassette of a gene of a nuclear-anchoring pro- tein that binds both to (i) a specific DNA sequence and (ii) to a suitable nuclear component and (B) a multimerized DNA binding sequence for said nuclear-anchoring protein, are capable of spreading in a proliferating cell population. Such nuclear- anchoring proteins include, but are not limited to, chromatin-anchoring proteins, such as the Bovine Papilloma Vims type 1 E2 protein (BPV1 E2). The DNA binding se- quences of these vectors were in one embodiment disclosed to be multimerized E2 binding sites.

A gene delivery vector system named Gene Transport Unit (GTU*) was disclosed in WO02/090558 which was particularly well suited for superior expression of therapeu- tic cDNAs (vaccines, antibodies, cytokines, etc.) in vivo. Compared to the regular mammalian expression vectors, the GTU* vector expresses E2 protein of bovine papillomavirus type 1 (BPV1): The E2 interacts specifically with many cellular factors (e.g. transcription machinery, mitotic chromatin) and is also bound to the GTU* plasmid itself through DNA-binding dimerization domain of the E2 protein and to the oligomer- ized E2 binding sites in the GTU* backbone.

Chemotherapeutic monoclonal antibodies

Rituximab was the first chemotherapeutic monoclonal antibody (CmAb) approved for clinical use in cancer therapeutics in 1997 and has significantly improved the clinical outcomes in non-Hodgkin's lymphoma. Since then, numerous CmAbs have been de- veloped and approved for the treatment of various haematologic and solid human cancers. Still, there is is a need for higher efficacy and significantly reduced toxicity of the today available CmAbs. Although great strides have been made in antibody engineering and cancer therapy, production cost is estimated at twice that required for conventional drugs. Further, production requires the use of very large cultures of cells, which are expensive to maintain. Gene therapy is a very promising treatment for many diseases, including monogenic diseases, cancer, cardiovascular disease, and neurodegenerative diseases. For cancer gene therapies to be increasingly successful, however, a major hurdle must be overcome: the development of gene delivery vectors that can safely, efficiently, and specifically deliver genetic material to the target cells.

Viral vectors are clearly suitable for both therapeutic application and as tools for biological studies; however, their delivery properties can be challenging to engineer and improve. The most frequently used have been based on adenovirus, retrovirus, vaccinia virus, herpesvirus, and AAV.

Barriers for viral vectors include: prior exposure of most people to natural virus leading to anti-virus neutralizing antibodies that can reduce vector delivery efficiency by orders of magnitude in vivo, poor vector biodistribution to important tissue targets, limited spread within those tissues, an inability to target specific cells, and limited effh ciency for many therapeutically relevant target cells. Also, the expression obtained by most vectors is short lived: the vectors tend to disappear from the transfected cells and are not transferred to daughter cells in a dividing cell population.

As a rule, the expression of therapeutic cDNAs is rapidly silenced by immunological mechanisms against the same vectors. Thus, for gene-based treatment, requiring higher and more prolonged expression of transgenes, new expression vectors have long been sought-after.

Astonishingly, the present invention for the first time discloses that GTU® expression vectors are particularly well suited for long-term repeated gene transfer treatments, such as for expressing proteins and/or fragments thereof binding an antigen associated with cancer, tumor(s) or solid tumor(s) and/ or cell signaling associated with said cancer, tumor(s) or solid tumor(s), i.e. that they are particularly well suited for delivery of transgenic CmAbs to cancer patients.

The present invention further for the first time discloses a new and further improved gtGTU® vector system tailored to be especially well-suited for gene-based cancer treatments, such as for delivery of transgenic CmAbs to cancer patients. In the new expression vectors disclosed herein, the ability of the expression vector to activate in- nate immune responses is even further reduced and thus the risk that the vector itself functions as a DNA vaccine is minimized, which potentially induces immunity against the product of interest, especially when administered repetitively.

SUMMARY OF THE INVENTION

The present invention relates to a non-integrating expression vector for use in a repeated administration regimen for gene therapy comprising: a DNA sequence encoding an E2 protein of Bovine Papilloma Virus type 1 (BPV1), comprising a DNA-binding dimerization domain, said E2 protein being operatively linked to a heterologous promoter, an olkjomerized DNA sequence, forming a binding site for an E2 protein of Bo- vine Papilloma Virus type 1 (BPV1). Said vector lacks an origin of replication functional in any mammalian cell, and said vector does not induce long-term adaptive immune responses in the patient treated with the gene therapy. Said vector further comprises one or more expression cassette(s) of a DNA sequence of interest being operatively linked to a heterologous promoter.

The heterologous promoters) in step a. and step c. can be the same promoter, two or more identical promoters, or two or more different promoters. One or all of the heterologous promoter(s) in step a. and/or step c. can optionally be (a) tissue specific promoters).

The present invention further relates to a non-integrating expression vector for use in a repeated administration regimen for gene therapy wherein said expression vector is further at least 50% devoid of immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter, and wherein at least 10% of the immunostimulatory sequences (ISSs) of said one or more expression cassette(s) of a DNA sequence of interest are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell. In some embodimnets, 50-100%, of all im- munostimulatory sequences (ISSs) from the plasmid backbone and from the promoter of said expression vector are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell, such as at least 10% of all human ISSs as shown in SEQ.ID.NO.: 16 and/or at least 10% of all mouse ISSs as shown in SEQ.ID.NO.: 17, in said expression cassette(s) of a DNA sequence of interest, are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell.

In a non-integrating expression vector for use according to the present invention at least 10% of all vector elements can further be minimized. Some embodiments of the present invention relate to a non-integrating expression vector for use according to the present invention, wherein said one or more expression cassette(s) of a DNA sequence of interest, encode(s) a protein or fragment(s) thereof binding an antigen associated with cancer, tumor(s) and/or solid tumor(s) and/or cell signaling associated with said cancer, tumor(s) and/or solid tumor(s). Said encoded protein or fragment(s) thereof binding an antigen can be selected from the group consisting of an antibody or fragment(s) thereof, a monoclonal antibody or fragment(s) thereof, and a protein or fragment(s) thereof which bind(s) an antigen via one or more epitope(s) associated with said cancer, tumor(s) and/or solid tumor(s) and/or cell signaling associated with said cancer, tumor(s) or solid tumor(s). Said anti- gen can be associated with one or more tumor(s) and/or solid tumor(s), with cell signaling affecting said cancer, tumor(s) and/or solid tumor(s), or be one or more signaling molecule(s) associated with said cell signaling affecting said cancer, tumor(s) and/or solid tumor(s). Said signaling molecule can be one or more growth factor(s), or one or more signaling molecule(s) associated with direct or indirect regulation of growth of said cancer, tumor(s) and/or solid tumor(s). Said encoded protein or fragments) thereof binding an antigen can inhibit said antigen activity, receptor activity and/or an interaction between said antigen and its one or more receptor(s). A non-integrating expression vector for use according to the present invention can directly or indirect regulate growth of said cancer, tumor(s) and/or solid tumor(s). This can e.g., but not limited to, be facilitated by regulating the supply of one or more nutrients) to said cancer, tumor(s) and/or solid tumor(s). For example, the present inven- tion in one embodiment relates to a non-integrating expression vector for use according to the present invention, wherein said antigen is a VEGF, preferably VEGF-1 and/or wherein said protein or fragment(s) thereof inhibits the interaction between VEGF and its receptor.

The present invention relates thus to a non-integrating expression vector for use according to the present invention, wherein said DNA sequence of interest encodes an anti-VEGF antibody or one or more fragment(s) thereof, comprising one or more of the CDR sequence(s) selected from the group consisting of the sequences listed in table 1.

Tabel 1:

In one embodiment, the present invention relates to a non-integrating expression vector for use according to the present invention, wherein said DNA sequence of interest encodes an anti-VEGF antibody or fragment(s) thereof, comprising one or more of the variable regions selected from the group consisting of:

and In one embodiment, the present invention relates to a non-integrating expression vector for use according to the present invention, wherein said DNA sequence of interest encodes an anti-VEGF antibody or fragment(s) thereof, comprising the variable re- gion:

In one embodiment, the present invention relates to a non-integrating expression vector for use according to the present invention, wherein said DNA sequence of interest encodes an anti-VEGF antibody or fragment(s) thereof, comprising the variable region:

In one embodiment, the present invention relates to a non-integrating expression vector for use according to the present invention, wherein the DNA sequence of interest in one or more expression cassette(s) encodes the bevacizumab DNA sequence op- eratively linked to a heterologous promoter.

In some embodiments, the present invention relates to a non-integrating expression vector for use according to the present invention, comprising at least one tissue specific heterologous promoter, wherein the tissue specific heterologous promoter is tissue specific for any one or more of the tissues or cells selected from the group consisting of: Langerhans cells, epidermis, keratinocytes, lung cells, muscle cells (includ- ing but not limited to smooth muscle, skeletal muscle and cardiac muscle cells), melanocytes, Merkel cells, liver cells (including but not limited to hepatocytes) , Langerhans cells, epithelial cells (including but not limited to alveolar type epithelial cells), endothelial cells, stroma cells and bone marrow cells (including but not limited to hematopoietic cells). In a presently preferred embodiment, said tissue specific heterologous promoter is tissue specific for Langerhans cells, epidermis, keratinocytes, muscle cells or liver cells. E.g. said tissue specific heterologous promoter can be selected from the group of promoters consisting of: : Dectin-2 (SEQ.ID.NO.: 22), Keratin 14 (SEQ.ID.NO.: 23), Keratin 10 (SEQ.ID.NO.: 24), Involucrin (SEQ.ID.NO.: 25), Loricrin (SEQ.ID.NO.: 26), Tni/3xUSE (SEQ.ID.NO.: 27) and MAP+AFP Ell

(SEQ.ID.NO.: 28).

A non-integrating expression vector for use according to the present invention can be a minicircle plasmid.

In some embodiments, the present invention relates to a non-integrating expression vector for use according to the present invention, wherein the DNA sequence of interest encodes a macromolecular drug. In one aspect, said DNA sequence of interest can encode the light chain of an antibody comprising and/or consisting of the follwoing sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAP- KVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA- TYYCQQYSTVPVvTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG- TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT- LSKADYEKHKVYACEVTHQGLSSPvTKSFNRGEC (SEQ.ID.NO.:37). In another aspect, said DNA sequence of interest can encode the light chain of an antibody comprising and/or consisting of the follwoing sequence: EVQLVESGGGLVQPGGSLRLS- CAASGYTFTNYGMNvWRQAPGKGLEvWGWINTY- TGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKY- PHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG- TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS- LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG- PSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAK- TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK- TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN- NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL- SPGK ( SEQ.ID.NO.:38). The present invention for the first time discloses a non-integrating expression vector for expressing a protein and/or fragment(s) thereof binding an antigen associated with cancer, tumor(s) or solid tumor(s) and/ or cell signaling associated with said cancer, tumor(s) or solid tumor(s) comprising: a DNA sequence encoding an E2 protein of Bo- vine Papilloma Virus type 1 (BPV1), comprising a DNA-binding dimerization domain, said E2 protein being operatively linked to a heterologous promoter, an oligomerized DNA sequence, forming a binding site for an E2 protein of Bovine Papilloma Virus type 1 (BPV1), wherein said vector lacks an origin of replication functional in any mammalian cell, wherein said vector does not induce long-term adaptive immune re- sponses in a patient treated with non-integrating gene therapy, and wherein said vector further comprises: one or more expression cassette(s) of a DNA sequence of interest being operatively linked to a heterologous promoter.

The expression vector according to the present invention comprises one or more het- erologous promoters), which can be the same promoter, two or more identical promoters, or two or more different promoters.

The one or more heterologous promoter(s) can be (a) tissue specific promoter(s). Some embodiments of the present invention relate to a non-integrating expression vector according to the present invention, wherein said vector is further at least 50% devoid of immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter, and wherein at least 10% of the immunostimulatory sequences (ISSs) of said one or more expression cassette(s) of a DNA sequence of interest are re- moved and/or modified in such a way that they do not activate an innate immune response in a mammalian cell.

The present invention further relates to a non-integrating expression vector according to the present invention, for use as a medicament, such as for use in gene therapy, such as for use in a repeated administration regimen for gene therapy, such as for use in a repeated administration regimen for non-integrating gene therapy.

Some embodiments of the present invention relate to a non-integrating expression vector according to the present invention, for use as a carrier vector for a gene, genes, or a DNA sequence or DNA sequences of interest, such as for one or more genes, or a DMA sequence or DNA sequences encoding a protein or peptide of an antibody, a therapeutic agent, a macromolecular drug, or any combination thereof.

Some embodiments of the present invention relate to a non-integrating expression vector according to the present invention, for a use selected from the group consisting of treating and/or preventing an inherited or acquired genetic defect, for use in antibody and/or monoclonal antibody therapy, for use in enzyme replacement therapy, for use in therapy with antibody or monoclonal antibody fragments, for use in delivery of an acute medicament, for use in cancer treatment, for production of a therapeutic macromolecular agent in vivo and for treatment of viral infections. The present invention in some embodiments thus relate to a non-integrating expression vector according to the present invention, for use in treating and/or preventing an inherited or acquired genetic defect; a condition which can be treated and/or prevented by antibody or mono-clonal antibody therapy; a condition which can be treated and/or prevented by enzyme replacement therapy; a condition which can be treated and/or prevented with antibody or monoclonal antibody fragments; a condition which can be treated and/or prevented by the delivery of an acute medicament; cancer; and/or a viral infection. The present invention in addition relates to a pharmaceutical composition comprising one or more expression vectors according to the present invention. Said pharmaceutical composition can comprise one or more additional pharmaceutically acceptable carrier(s). Typically, said pharmaceutically acceptable carrier is selected from the group consisting of: buffer, antimicrobial agents, antioxidants, tonicity agents, water, bulking agents, wetting agents and surfactants.

The invention further relates to a pharmaceutical composition according to the present invention for use as an injectable medicament, in particular for use in a repeated administration regimen for gene therapy, such as for use in a a repeated administration regimen for non-integrating gene therapy.

In consequence, the present invention also relates to the use of a non-integrating expression vector and/or a pharmaceutical composition according to the present invention for the manufacture of a medicament for use in a repeated administration regimen for gene therapy, such as for use in a repeated administration regimen for non- integrating gene therapy. In combination and/or in addition the present invention relates to the use of a non-integrating expression vector and/or a pharmaceutical composition according to the present invention for the manufacture of a medicament for use in treating and/or preventing an inherited or acquired genetic defect, for use in antibody or monoclonal antibody therapy, for use in therapy with antibody or monoclonal antibody fragments, for use in enzyme replacement therapy, for use in delivery of an acute medicament, for use in cancer treatment, for production of a therapeutic mac- romolecular agent in vivo, and/or for the treatment of infectious diseases, such as a viral infection. The present invention thus relates to the use of a non-integrating expression vector and/or a pharmaceutical composition according to the present invention for the manufacture of a medicament for use in treating and/or preventing an inherited or acquired genetic defect; a condition which can be treated and/or prevented by antibody or mono-clonal antibody therapy; a condition which can be treated and/or prevented by enzyme replacement therapy; a condition which can be treated and/or prevented with antibody or monoclonal antibody fragments; a condition which can be treated and/or prevented by the delivery of an acute medicament; cancer; and/or a viral infection. In one aspect, the present invention further relates to a method of treatment comprising repeated, long term and/or life-long non-integrating gene therapy, for treating an inherited or acquired genetic defect, for antibody or monoclonal antibody therapy, for use in therapy with antibody or monoclonal antibody fragments for enzyme replacement therapy, for cancer treatment, for treating a tumor or solid tumor, for delivery of an acute medicament, for production of a therapeutic macromolecular agent in vivo and/or for treatment of viral infections in a subject in need of said treatment, said method comprising: administering to said subject a therapeutically effective amount of one or more expression vector(s) and/or pharmaceutical composition(s) according to the present invention, wherein said vector comprises a DNA sequence of interest which encodes a protein or fragment(s) thereof which is affected by said inherited or acquired genetic defect, such as but not limitted to leading to cancer, tumor(s) or solid tumor(s), and/or which binds an antigen that is associated with cancer, tumor(s) or solid tumor(s). The present invention thus relates to relates to a method of treatment comprising repeated, long term and/or life-long non-integrating gene therapy, the use of a non-integrating expression vector and/or a pharmaceutical composition according to the present invention for the manufacture of a medicament for use in treating and/or preventing an inherited or acquired genetic defect; a condition which can be treated and/or prevented by antibody or mono-clonal antibody therapy; a condition which can be treated and/or prevented by enzyme replacement therapy; a condition which can be treated and/or prevented with antibody or monoclonal antibody fragments; a condition which can be treated and/or prevented by the delivery of an acute medicament; cancer; and/or a viral infection, said method comprising: administering to said subject a therapeutically effective amount of one or more expression vector(s) and/or pharmaceutical composition(s) according to the present invention, wherein said vector comprises a DMA sequence of interest which encodes a protein or fragments) thereof which is affected by said inherited or acquired genetic defect, such as but not limited to leading to cancer, tumor(s) or solid tumor(s), and/or which binds an antigen that is associated with cancer, tumor(s) or solid tumor(s).

In a method of treatment according to the present invention, said one or more expression vector(s) express(es) a light chain of an antibody and/or a heavy chain of the same antibody. Said one or more expression vector(s) expressing said light and/or heavy chain is/are administered to said subject in any one of the following ways: a. both expression vectors are injected at the same time b. the expression vector encoding said light chain is administered first, followed by the expression vector encoding said heavy chain c. the expression vector encoding said heavy chain is administered first, followed by the expression vector encoding said light chain

Said one or more expression vector(s) is/are typically administered in the form of a pharmaceutical composition according to the present invention. FIGURES

Figure 1

Usage of the GTU* for expression of therapeutic products in vivo.

Schematic gene delivery vector system named Gene Transport Unit (GTU*) for supe- nor expression of therapeutic cDNAs (vaccines, antibodies, cytokines, etc.) in vivo. Compared to the regular mammalian expression vectors, the GTU* expresses E2 protein of bovine papillomavirus type 1 (BPV1): The E2 interacts specifically with many cellular factors (e.g. transcription machinery, mitotic chromatin) and is also bound to GTU® plasmid itself through DNA-binding dimerization domain of the E2 protein and to the oligomerized E2 binding sites in the GTU* backbone.

Figure 2

Expression in epidermis: GTU* vs "non-GTU"

Figure 3

Expression in epidermis: GTU* vs "non-GTU"

Figure 4

Expression in epidermis: GTU* vs "non-GTU"

Figure 5

Expression in epidermis: GTU* vs "non-GTU"

Figure 6

SEAP Expression in muscle: EP using ichor device

Figure 7

LUCIFERASE Expression from GTU* in muscle: HYALuriniDASE enhanced in EP GTU® and non-GTU® control vectors were designed and constructed. All vectors contained tricistronic GEF markergene expressing three marker proteins from single cDNA:

1. secretable Gaussia luciferase (Glue) measurable in blood samples;

2. EGFP detectable by fluorescence using in vivo imaging;

3. intracellular firefly luciferase that can be measured in muscle cell extracts or by in vivo imaging with luciferine.

Figure 8

Expression of mouse antibody in mouse muscle from the GTU*

Instead of surrogate marker, actual potential therapeutic molecule (antibody) is expressed as gene of interest from the GTU® or the non-GTU® control. Six vectors expressing intact mouse lgG2a antibody 5c2 against human (and also mouse) MANF protein are shown. The expression from the delivered plasmids is quantitatively ana- lysed by MANF ELISA in peripheral blood. For sampling, 1 : 20 diluted blood samples were collected at different time points by mixing 5 μΙ of blood from tale tip cut with 95 μΙ of PBS. The analysis time points were collected in the next time-points: 0 (baseline before DNA delivery), 3 days, 1, 2, 3, 4, 5, 6, 8 and 12 weeks. All time points were analysed by pooling the samples (dilution 1:160) in the group.

Figure 9

Expression of mouse antibody in mouse muscle from the GTU*

Scatter blots of the individual samples in time-points 4, 6, 8, and 12 weeks. Statistically significant differences between GTU® and regular group are shown by asterisks. Figure 10

Antibody production in muscle from from GTU* reaches to therapeutic concentrations in mouse. Lower diagram (muscle GTU): in time data of individual animal of the same set as above (3xUSE/Tnl controlling both the E2 and antibody expression). anti-MANF MAb concentration in the blood was quantified in the peak level. Upper diagram: Same but "ubiquitous GTU" was delivered. In this GTU® the E2 expression was controlled by the 3xUSE/Tnl but antibody expression was under control of strong ubiquitous CAG promoter.

Figure 11

The gtGTU® concept

Figure 12

Schemes of three gtGTU® configurations designed. Coding sequences for Glue and E2 are indicated by red colour.

Figure 13

Glue activities in supernatants of RD cells transfected with equimolar amounts of pMA-gtGTU1-Gluc or with control plasmids. RD cells were transfected by electro- poration (0.4 cm cuvettes, 190 V, 975 pF) with different amounts (100, 250 and 1000 ng) of pMA-gtGTU1-Gluc expressing the Glue markergene. In comparison, equimolar amounts of gene vaccination plasmids GTU8-moptGluc (90, 225 and 900 ng) and pCMV-moptGluc (50, 125 and 500 ng) were transfected. Twenty hours and 44h post- transfection Glue activity was measured from the 5 μΙ of culture medium using Bio- Lux ⁸ Gaussia Luciferase Assay Kit (New England Biolabs) and Glomax 20/20 lumi- nometer (Promega) according to manufacturers' instructions, riu - relative light unit. Figure 14

Western blot analysis of E2 expression from GTU8-moptGluc and pMA-gtGTU1. RD cells were transfected as described in the legend of Figure 3. The cells were lysed at 44h post-transfection with Laemli lysis solution + 100 mM DTT. The expression of E2 was analysed by Western blot using cocktail of mouse monoclonal antibodies against E2 as primary antibody and goat anti mouse IgG conjugated with HRP as secondary antibody (Labas). The signals were visualised by ECL Plus Western blotting detection system (Amersham). The size (calculated by aa sequence) of E2 (position also indicated) is 45 kD.

Figure 15

Plasmids used in the experiments, results of which are shown in figures 2-4.

Figure 16

Plasmids used in the experiments, results of which are shown in figure 5.

Figure 17

Plasmids used in the experiments, results of which are shown in figure 7.

Figure 18

Plasmids used in the experiments, results of which are shown in figure 10.

Figure 19

Plasmids used in the experiments, results of which are shown in figures 13 and 14. Figure 20

Comparison of sequences of functional regions in GTU8-Gluc vs gtGTLH-Gluc.

SEQ.ID.NO. 1: template 1, SEQ.ID.NO. 2: template 2.

Figure 21

Map of representative gtGTU® minicircle, SEQ.ID.NO.:3.

Figure 22

Map of representative gtGTU® minicircle, SEQ.ID.NO.:4.

Figure 23

Map of representative gtGTU® minicircle, SEQ.ID.NO.:5

Figure 24

Schematic maps of the vectors used in Example 5. Fig. 24a: SEQ.ID.NO.: 6, Fig. 24b: SEQ.ID.NO.: 7, Fig. 24c: SEQ.ID.NO.: 8, Fig. 24d: SEQ.ID.NO.: 9, Fig. 24e:

SEQ.ID.NO.: 10, Fig. 24f: SEQ.ID.NO.: 11, Fig. 24g: SEQ.ID.NO.: 12, Fig. 24h:

SEQ.ID.NO.: 13, Fig. 24h: SEQ.ID.NO.: 14, Fig. 24 i: SEQ.ID.NO.: 15.

Figure 25

a) Timeline of DNA delivery used used in Example 5; b) Delivery of DNAs by EP as well as i.p. administration of the proteins, collection of blood samples, tumor induction, measurements and collection are indicated.

Figure 26 Tumor growth curves in mice which received the A.4.6.1. or 8E2 MAb protein via i.p. route.

Figure 27

Tumor growth curves in mice which received the A.4.6.1. or 8E2 MAb expressing DMA via i.m. route. G1-G4 are sorted using color codes representing different treatment groups.

Figure 28

Antibody production in animals treated with DMA vectors.

Figure 29

Survival data for female mice received the A.4.6.1. and 8E2 MAb via i.m. DNA delivery.

Figure 30

Percentage of animals belonging to clusters (in Fig. 31) showed tumor inhibition at day 21 and 30 days after injection of A673 cells, respectively.

Figure 31

Tumor growth rates (% of growth) were analyzed in 2. week (days 7-14) and 3. week (days14-21) after injection of the A673 cells. Statistically significant differences are indicated by asterisks and p values.

Figure 32

A.4.6.1. blood concentration kinetics up to 8 week after DNA delivery.

Figure 33

A.4.6.1. MAb levels of individual animals in 5, 6 and 8 week time-points.

Figure 34

mlL-6 standard

Figure 35

mTNF-a standard

Figure 36

IL6 concentrations

Figure 37

TNFa concentrations

Figure 38

mlL-6 standard

Figure 39

mTNF-a standard

Figure 40 IL6 concentrations

Figure 41

TNFa concentrations

Figure 42

Schematic maps of GTIKS ⁾ and gtGTU® plasmids gtGTUM(i)-nult#27 and GTUM(i)- null#89 used in example7.

Figure 43

Immunization and sample collection timeline example 7.

Figure 44

OVA specific lgG1 readouts of pooled samples in different time-points

Figure 45

OVA specific lgG2a readouts of pooled samples in different time-points

Figure 46

OVA specific lgG1 response in time (OVA ELISA, blood dilution 1:800)

Figure 47

OVA specific lgG1 response in time (OVA ELISA, blood dilution 1:800)

Figure 48

OVA specific lgG1 and lgG2a responses in 22-day time-point (OVA ELISA, blood dilution 1:1000).

DET AILED DESCRIPTION OF THE INVENTION

The present invention for the first time discloses a new and surprising use of GTU® expression vectors for expressing protein or fragment(s) thereof binding an antigen associated with cancer, tumor(s) or solid tumor(s) and/or cell signaling associated with said cancer, tumor(s) or solid tumor(s), such as for delivery of transgenic CmAbs to cancer patients and thus for treating cancer, as well as an improved expression vector system hereafter referred to as gtGTU® expression vectors for use in expressing protein or fragment(s) thereof binding an antigen associated with cancer, tumor(s) or solid tumor(s) and/ or cell signaling associated with said cancer, tumor(s) or solid tu- mor(s), such as for delivery of transgenic CmAbs to cancer patients for treating cancer.

In the present context the term "repeated administration regimen" is used to describe admininstration regimens wherein the patient treated receives more than a single dose of the vector genome/expression vector, such as at least 2, 3, 5, 10, 20, 100 or 200 doses of vector genome/expression vector. In some embodiments, the term is used to describe a weekly or biweekly or monthly or yearly administration of the vector genome/expression vector. The frequency and/or duration of the repeated administration regimen is typically adjusted after evaluation of effectivity of the treatment and will vary depending on the disease to be treated, the patients response to the treatment, as well as the nature of the protein and/or fragment of protein encoded in the expression cassette(s) of the expression vector administered.

The expression vector(s) of the present invention is/are particularly well suited for a repeated administration in a gene therapy regimen because they do not activate an immunological response in the patient treated with the gene therapy, which prevents the development of adaptive immune responses for long term efficacy. The expression vector(s) of the present invention do(es) not activate an innate immune response in a mammalian cell, as is demonstrated in the experimental section, wherein in par- ticular experiments 6 and 7 clearly disclose that the expression vectors) of the present invention do(es) not induce humoral responses in an animal model. It is therefore concluded that the expression vector(s) of the present invention will not activate a significant induction of antigen specific lgG1 response in the patient treated with the gene therapy. The new expression vectors achieve substantially higher and more prolonged expression of transgenes compared to traditional gene expression vectors, such as vectors built on a GTU* backbone. In consequence, the new expression vectors are particu- larly well suited for gene-therapy treatment, as they need only be administered at more extended intervals than is necessary with traditional gene expression vectors.

The new expression vectors are in particular suitable for gene-based treatments and/or therapies, wherein an expression of a therapeutic magnitude and moderate du- rabilrty is desired, reducing potential adverse effects of the mAb in the patient, e.g. when expressing mAb in cancer therapy, such as in particular in passive immunotherapy for cancer and/or in the treatment of viral infections.

In each of the new expression vectors, a bulk of immunostimulatory sequences (ISSs) are removed, thus reducing the ability of the expression vectors to activate innate immune responses and thus decreasing the risk that the vector itself functions as a DNA vaccine, which can potentially induce immunity against the gene-product of interest, especially when administered repetitively. Optimizing the magnitude and durability of gene expression will allow for longer intervals between administrations, effectively re- ducing the need for repetitive administration in total.

The new expression vector system is further improved by minimizing the size of the plasmid vector and removal of dispensable components (e.g. bacterial origin, antibiotic resistance marker), thus further improving the vector outcome in gene therapy applications in vivo.

For the design and construction of the herein for the first time presented gtGTU® expression vectors, whole sequences of the prior disclosed GTLT* gene vaccination vector (GTU8) were completely redesigned to avoid mouse and/or human ISSs. In addi- tion, in one presently preferred embodiment, a gtGTU® expression vector is produced in a particularly compact form as a minicircle plasmid.

In essence, the present invention discloses several novel expression vectors with reduced ability to activate an innate immune response in a mammalian cell, which meets the requirements of a carrier vector of a gene and/or genes of interest for improved gene therapy.

A typical expression vector of the present invention is a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell comprising (a) a DNA sequence encoding a nuclear-anchoring protein operatively linked to a heterologous promoter, said nuclear-anchoring protein comprising (i) a DNA binding domain which binds to a specific DNA sequence, and (ii) a functional domain that binds to a nuclear component, or a functional equivalent thereof; and (b) a multimerized DNA sequence forming a binding site for the nuclear anchoring protein, wherein said vector lacks an origin of replication functional in any mammalian cell, and wherein said vector is at least 50% devoid of immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter, and wherein at least 10% of the immunostimulatory sequences (ISSs) of said one or more expression cassette(s) of a DNA sequence of interest are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell.

In one embodiment, a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell comprises (a) a DNA sequence encoding an ΕΞ2 protein of Bovine Papilloma Virus type 1 (BPV1), comprising a DNA- binding dimerization domain, said E2 protein being operatively linked to a heterologous promoter, and (b) an oligomerized DNA sequence, forming a binding site for an E2 protein of Bovine Papilloma Virus type 1 (BPV1), wherein said vector lacks an origin of replication functional in any mammalian cell, and wherein said vector is at least 50% devoid of immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter, and wherein at least 10% of the immunostimulatory sequences (ISSs) of said one or more expression cassette(s) of a DNA sequence of interest are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell.

A non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention is a vector in which at least 50%, preferably 50-100%, of all immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell, pref- erably so that they do not activate an innate immune response in any mammalian cell, e.g. in any cell selected from any murine or human cells. Said vector is thus at least 50% devoid of immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter.

In one embodiment, a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention is a vector wherein at least 10%, such as 10-100%, preferably 50-100% of all vector elements are minimized. Said expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention can e.g. be a minicircle plasmid.

A non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention can further comprise one or more expression cassette(s) of a DNA sequence of interest.

In one embodiment, a non-integrating expression vector according to the present invention comprises one or more expression cassette(s) of a DNA sequence encoding at least one of

a. a heavy chain and

b. a light chain, of

a tumour specific antibody.

In particular, the tumour specific antibody can be an angiogenesis inhibitor, such as selected from the group consisting of A.4.6.1 and Bevacizumab.

In another embodiment, a non-integrating expression vector according to the present invention comprises one or more expression cassette(s) of a DNA sequence encoding at least one of

a. a heavy chain and

b. a light chain, of

a virus specific antibody.

Said one or more expression cassette(s) of a DNA sequence of interest typically codes for a non-immunogenic intrinsic protein. A non-immunogenic intrinsic protein can be a naturally non-immunogenic intrinsic protein, or a protein, wherein at least 10%, such as at least 10-100%, preferably 50- 100% of all immunostimulatory sequences (ISSs) of said one or more expression cas- sette(s) of a DMA sequence of interest are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell.

Typically, a non-integrating expression vector according to according to the present invention comprises one or more expression cassette(s) of a DNA sequence of inter- est, wherein at least 10% of all human ISSs as shown in SEQ.ID.NO.: 16 and/or at least 10% of all mouse ISSs as shown in SEQ.ID.NO.: 17 are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell. A non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention can comprise a heterologous promoter which is ubiquitous and/or a heterologous promoter which is tissue specific. In one embodiment, a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell, according to the present invention, comprises a heterologous promoter which is tissue specific for any one or more of the group of tissues or cells consisting of Langerhans cells, epidermis, keratino- cytes, lung cells, muscle cells (including but not limited to smooth muscle, skeletal muscle and cardiac muscle cells), melanocytes, Merkel cells, liver cells (including but not limited to hepatocytes) , Langerhans cells, epithelial cells (including but not limited to alveolar type epithelial cells), endothelial cells, stroma cells, bone marrow cells (including but not limited to hematopoietic cells). Examples of vectors of the present invention are shown in figures 11 , 12, 15, 16, 17, 18, 19, 21 , 22, 23, and 24. Figure 20 gives the nucleic acid sequence of one vector of the present invention as exemplified by gtGTU1-Gluc. It is in general understood that any nucleic acid sequence which is at least 90, such as 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% identical to any of the nucleic acid sequences exemplified herein is still considered to be a nucleic acid sequence according to the present invention. In one aspect, the present invention relates to a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention, wherein the DNA sequence of interest encodes a protein that is defective in a hereditary single gene disease.

Furthermore, a specific embodiment of the present invention is e.g. an ISS-free promoter corresponding to a nucleic acid sequence selected from any one of the nucleic acid sequences as shown or represented as elements of a plasmid in figures 11 , 12, 15, 16, 17, 18, 19, 21, 22, 23, or 24, as well as its use in producing a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention.

A specific embodiment of the present invention is also an ISS-free polyadenylation signal corresponding to any one of the nucleic acid sequences selected from any one of the nucleic acid sequences as shown or represented as elements of a plasmid in figures 11, 12, 15, 16, 17, 18, 19, 21 , 22, 23, or 24, and for use in producing a non- integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention.

Another specific embodiment of the present invention is a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention as shown in SEQ.ID.NO.: 2 or any nucleic acid sequence which is at least 90, such as 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% identi- cal to SEQ.ID.NO.: 2.

A hereditary single gene disease can in the present context relate to a disease selected from the group consisting of Huntington disease, Marfan syndrome, neurofibromatosis, retinoblastoma, Polydactyly, phenylketonuria, cystic fibrosis, sickle cell anemia, Tay-Sachs disease, hemophilia A, Duchenne muscular dystrophy, glucose-6- phosphate dehydrogenase deficiency, Rett syndrome, severe combined immunodeficiency, Hemophilia B and Alpha-1 Antitrypsin Deficiency.

In one aspect, the present invention relates to a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell ac- cording to the present invention, wherein the DNA sequence of interest encodes a protein associated with immune maturation, regulation of immune responses, regulation of autoimmune responses, a macromolecular drug, a pathogen specific antibody, a tumor specific antibody, a virus specific antibody and/or an enzyme for enzyme re- placement therapy.

The present invention in particular relates to a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention, for use as a medicament, such as for use as a carrier vector for a gene, genes, or a DMA sequence of interest, a DNA sequences encoding a protein or peptide of an antibody, a therapeutic agent, a macromolecular drug, or any combination thereof, and/or for use in gene therapy.

A non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention is typically intended for a use selected from the group consisting of; treating and/or preventing an inherited or acquired genetic defect, for use in monoclonal antibody therapy, for use in enzyme replacement therapy, for use in delivery of an acute medicament, for use in cancer treatment, for production of a therapeutic macromolecular agent in vivo and for treat- ment of a viral infection(s).

In particular, a non-integrating expression vector according to the present invention is intended for use in cancer treatment, such as, but not limited to, use in passive immunotherapy for cancer, e.g.in MAB based immunotherapy for cancer.

In one embodiment, the present invention thus relates to a gene therapeutic agent comprising and/or consisting of and/or containing a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell, according to the present invention.

The present invention further relates to the use of a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell, according to the present invention, for the manufacture of a medicament for treating and/or preventing an inherited or acquired genetic defect, for use in monoclonal anti- body therapy, for use in enzyme replacement therapy, for use in delivery of an acute medicament, for use in cancer treatment, for production of a therapeutic macromolec- ular agent in vivo, and/or for treatment of a viral infection(s).

Also related to is a method for treating an inherited or acquired genetic defect, for monoclonal antibody therapy, for enzyme replacement therapy, for cancer treatment, for delivery of an acute medicament, for production of a therapeutic macromolecular agent in vivo and/or for treatment of a viral infection(s) in a subject in need of said treatment, said method comprising: administering to said subject a therapeutically effective amount of a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention, wherein said vector comprises a DMA sequence of interest which encodes a protein which is affected by said inherited or acquired genetic defect, or which is effective in said inherited or acquired genetic defect or in said monoclonal antibody therapy, enzyme replacement therapy, cancer treatment, delivery of an acute medicament, and/or treatment of a viral infection(s).

Further envisioned is a method for producing a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell, according to the present invention, comprising removing and/or altering at least 50%, preferably 50-100%, of all immunostimulatory sequences (ISSs) of the expression vector by intelligent mutagenesis.

A vector according to the present invention can be produced by selecting a DNA sequence encoding an E2 protein of Bovine Papilloma Virus type 1 (BPV1) comprising a DMA-binding dimerization domain, said E2 protein being operatively linked to a heterologous promoter, and an oligomerized DNA sequence, forming a binding site for an E2 protein of Bovine Papilloma Virus type 1 (BPV1), wherein said vector lacks a papilloma virus origin of replication functional in any mammalian cell, and removing and/or modifying at least 50% of all immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter by intelligent mutagenesis, so that they do not activate an innate immune response in a mammalian cell, and alternatively in addition minimizing at least 10-100% of all vector elements and/or removing and/or modifying at least 10% of all human ISSs as shown in SEQ.ID.NO.: 16 and/or at least 10% of all mouse ISSs as shown in SEQ.ID.NO.: 17, in said expression cassette(s) of a DNA sequence of interest, in such a way that they do not activate an innate immune re- sponse in a mammalian cell. Said method can further comprise cultivating a host cell containing said expression vector; and recovering the expression vector, and/or before this, transforming said host cell with said vector, wherein said host cell is a pro- karyotic or a eukaryotic cell, such as but not limited to an Escherichia coli cell.

In one embodiment, all human ISSs (TCGTW (SEQ.ID.NO.: 16)) and mouse ISSs (RRCGYY (SEQ.ID.NO.: 17)) are in said expression cassette(s) of a DNA sequence of interest removed by point mutations. A host cell is herein also disclosed, characterized by containing a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell, according to the present invention. Said host cell is typically a bacterial cell, or a mammalian cell. Said host cell is a prokaryotic or a eukaryotic cell, such as but not limited to an Escherichia coli cell.

Consequently, the present invention also relates to a pharmaceutical composition comprising a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell according to the present invention, and a suitable pharmaceutical carrier.

VECTORS OF THE INVENTION

The vectors of the present invention lack an origin of replication functional in any mammalian cell and they do not induce long-term adaptive immune responses in a patient treated with them.

The expression vectors) of the present invention are non-integrating viral expression vectors which do not induce innate immune responses in the patients treated and are therefore surprisingly well suited for use in a repeated administration regimen for non- intergrating gene therapy.

The presently disclosed non-integrating expression vectors are particularly well suited for long term and/or life long treatment with non-integrating gene therapies because they do not induce long-term adaptive immune responses in the patients treated. The expression vectors) of the present invention do not activate an immunological response in the patient treated with the gene therapy, which prevents the development of adaptive immune responses for long term efficacy. Experiments 6 and 7 clearly disclose that the expression vector(s) of the present invention do(es) not in- duce humoral responses in an animal model. It is therefore concluded that the expression vector(s) of the present invention will not activate a significant induction of antigen specific lgG1 response in the patient treated with the gene therapy.

A non-integrating expression vector for use in a repeated administration regimen for gene therapy, according to the the present invention, comprises (a) a DNA sequence encoding a nuclear-anchoring protein operatively linked to a heterologous promoter, said nuclear-anchoring protein comprising (i) a DNA binding do main which binds to a specific DNA sequence, and (ii) a functional domain that binds to a nuclear component, or a functional equivalent thereof; and (b) a multimerized DNA sequence form- ing a binding site for the nuclear anchoring protein as well as one or more expression cassette(s) of a DNA sequence of interest operatively linked to a heterologous promoter.

In some embodiments, the present invention relates to a novel and improved expres- sion vector comprising (a) a DNA sequence encoding a nuclear-anchoring protein operatively linked to a heterologous promoter, said nuclear-anchoring protein comprising (i) a DNA binding do-main which binds to a specific DNA sequence, and (ii) a functional domain that binds to a nuclear component, or a functional equivalent thereof; and (b) a multimerized DNA sequence forming a binding site for the nuclear anchoring protein, wherein said vector lacks an origin of replication functional in any mammalian cell, and which is at least 50%, such as at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100%, such as 10-100%, 10-20%, 20-50%, 30-60%, 50-75%, 75-100% or preferably 50-100% devoid of of all immunostimulatory sequences (ISSs) from the plasmid backbone and/or from the promoter.

The term "devoid" is in the present context employed to describe a percentage of ISSs in a given nucleic acid sequence, wich is lesser than the original % of ISSs in the nucleic acid sequence. Typically, this is a result from when known ISSs in a given nucleic acid sequence are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell, and thus the overall immunostimula- tory profile of the plasmid DNA is substantially reduced.

The ISSs so removed or modified are potentially immunostimulatory, i.e. they are the- oretically immunostimulatory according to the knowledge of the person skilled in the art.

ISS removal and/or modification reduces the ability of activation of innate immune responses and thus decreases the risk of the vector itself to work as a DNA vaccine which potentially can induce immunity against the product of interest, especially when administered repetitively;

The term ¹¹ removing" and/or .modifying" immunostimulatory sequences (ISSs) in the present context relates to manipulation of DNA sequences in order to reduce the im- munostimulatory profile of the plasmid DNA.

For example, point mutations wherein single nucleotide bases are changed, inserted or deleted in the DNA vector may be performed to remove and/or modify immunostimulatory sequences (ISSs).

Further, fragments of DNA may be synthetically made and introduced into the DNA vector, e.g. by polymerase chain reaction (PCR), to reduce the percentage of immunostimulatory sequences (ISSs). In one embodiment, the dinucleotide sequence CpG may be altered by site directed mutagenesis, either by removing cytosine or guanine or both, while maintaining the amino acid coding sequence to remove and or modify immunostimulatory sequences (ISSs). In a yet another embodiment, synthetic DNA fragments which are made deficient in the CpG sequence while maintaining the amino acid coding sequence are introduced by polymerase chain reaction (PCR) to remove immunostimulatory sequences (ISSs). ln one embodiment, nucleotides immediately flanking the CpG dinucleotides in the DNA vector may be altered by point mutagenesis to remove immunostimulatory sequences (ISSs) For example, the DNA sequence motif, GACGTT, when immediately flanking CpG dinucleotides, may be altered by point mutagenesis or by introducing synthetic DNA fragments by polymerase chain reaction (PCR) while maintaining the amino acid coding sequence may be performed to remove immunostimulatory sequences (ISSs). In another example, in adition, at least 10% of all human ISSs as shown in

SEQ.ID.NO.: 16 and/or at least 10% of all mouse ISSs as shown in SEQ.ID.NO.: 17, in a given expression cassette(s) of a DNA sequence of interest, are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell.

Intelligent mutagenesis

In the present context, the term intelligent mutagenesis is used to describe the process of introducing mutations, which are done such way that they do not hamper the functionality of the vector.

In general, in coding sequences, the ISSs removing and/or altering mutations are done in such a way that these do not create: (i) AU-rich RNA instability elements (like AUUUA); (ii) secondary RNA structures (e.g. hairpins); or rare codons for human or mouse cells.

The mutations are optionally, but presently preferably, also made in promoter and polyadenylation regions, as well as in introns. The sequences in these regions contain structural elements and binding sites for cellular factors that are usually important for transcription of the gene of interest and e.g. E2.

Generally, in the process of ISSs removal and/or modification, all crucial elements (including but not limited to TATA, CAAT, Inr, splice donor, splice acceptor, pA signals etc.) are kept intact. However, e.g. the CMV promoter contains five 19-bp elements that may be important for transcription. Four of them are identical and recognized as cAMP response elements (CRE) but contain mouse ISS in their core. The fifth 19-bp element is a variant of CRE (AF-1/CRE) and it does not contain ISS (Keller et a/., J Virol 77, 6666). Thus in one embodiment, all four ISS-containing 19-bp elements are replaced with AF-1/CRE elements in the gtGTUI vector. These changes do not hamper the activity of the promoter.

In consequence, in one embodiment, in the process of ISSs removal and/or amendment, any and/or all crucial element(s) (including but not limited to TATA, CAAT, Inr, splice donor, splice acceptor, pA signals etc.) are replaced by functionally identical or similar ISS-free, or at least ISS-reduced, alternative elements.

Site-directed mutagenesis to introduce site-specific mutations in DNA sequences may be performed by a large number of methods. The methods include but are not limited to for example Kunkel ^' s method, where the DNA to be mutated is inserted into a phagemid such as M13mp18/19 and is then transformed into an E.coli strain deificent in two enzymes, dUTPase (dut) and uracil deglycosidae (ung). Both enzymes are part of a DNA repair pathway that protects the bacterial chromosome from mutations by the spontaneous deamination of dCTP to dUTP. The dUTPase deficiency prevents the breakdown of dUTP, resulting in a high level of dUTP in the cell. The uracil degly- cosidase deficiency prevents the removal of uracil from newly synthesized DNA. As the double-mutant E. coli replicates the phage DNA, its enzymatic machinery may, therefore, misincorporate dUTP instead of dTTP, resulting in single-strand DNA that contains some uracils (ssUDNA). The ssUDNA is extracted from the bacteriophage that is released into the medium, and then used as template for mutagenesis. An oligonucleotide containing the desired mutation is used for primer extension. The heter- oduplex DNA, that forms, consists of one parental non-mutated strand containing dUTP and a mutated strand containing dTTP. The DNA is then transformed into an E. coli strain carrying the wildtype dut and ung genes. Here, the uracil-containing parental DNA strand is degraded, so that nearly the entire resulting DNA consists of the mutated strand.

A further example is cassette mutagenesis wherein a DAN fragment is synthesized, and inserted into the plasmid DNA which is then followed by cleavage using a restriction enzyme at a site in the plasmid and subsequent ligation of a pair of complementary oligonucleotides containing the mutation in the gene of interest to the plas- mid. Usually, the restriction enzymes that cut at the plasmid and the oligonucleotide are the same, permitting sticky ends of the plasmid and insert to ligate to one another. This method can generate mutants at close to 100% efficiency, but is limited by the availability of suitable restriction sites flanking the site that is to be mutated. Further, the polymerase chain reaction (PCR) may be utilized such that a fragment may be generated covering the desired mutation and two convenient restriction enzymes sites. The thus generated fragment may then be ligated into a cleaved vector and thereby introducing the point mutation. In addition, the CRISPR/Cas system can been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. In one embodiment, removing a bulk of immunostimulatory sequences (ISSs) from the GTU* DMA sequences is done by intelligent mutagenesis.

The present invention in one aspect relates to ISS-free variants of promoters (based on RSV LTR, CMV, for example), polyadenylation signals, introns etc. In particular but not exclusively intended for usage in gtGTU® vectors.

In general, gtGTU® vectors are designed based on minimizing the plasmid vector size by removal of dispensable components (e.g. bacterial origin, antibiotic resistance marker) which in itself improves the vector outcome in gene therapy applications in vivo. Higher and more prolonged expression of the transgene can be achieved. Further, the bulk of immunostimulatory sequences (ISSs), i.e. at least 50% of all ISSs, preferably at least 55% of all ISSs, are also removed, striving to completely remove all ISSs in gene therapy vectors. This reduces the ability of activation of innate immune responses and thus decreases the risk of the vector itself to work as a DNA vaccine which potentially can induce immunity against the product of interest, especially when administered repetitively, thus, minimizing the overall immunostimulatory profile of the plasmid DNA.

The present design includes minimizing the vector elements, e.g. by incorporating the E2BSs into the intron, and/or using miniplasmid format by removal of dispensable components, e.g. bacterial origin, antibiotic resistance marker. The removal of dispensable component e.g. further ensures more efficient delivery/expression.

In the present context, the term miniplasmid format is used to describe a plasmid with reduced size due to partial or complete removal of the elements needed for propagation in bacterial cells but not for its full functionality in eukaryotic cells.

In consequence, the present invention discloses a non-integrating expression vector, wherein at least 10%, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100%, such as 10-100%, 10- 20%, 20-50%, 30-60%, 50-75%, 75-100% or preferably 50-100%, of all vector elements are minimized. In a presently preferred embodiment, at least 50% of all vector elements are minimized. A presently preferred embodiment relates to a non-integrating expression vector, which is a minicircle plasmid.

In the present context, the term minicircle plasmid is employed to episomal DNA vectors that are produced as circular expression cassettes devoid of any bacterial plas- mid DNA backbone.

In one embodiment, a minicircle plasmid is a small circular plasmid devoid of prokary- otic vector elements and derived from a parental and bacterial plasmid with eukaryotic inserts.

The expression vector according to the present invention can further comprise one or more expression cassette(s) of a DNA sequence of interest, which code(s) for one or more non-immunogenic intrinsic protein(s), and/or which code(s) for one or more pro- tein(s), wherein at least 10%, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, such as 10- 100%, 10-20%, 20-50%, 30-60%, 50-75%, 75-100% or preferably 50-100%, of all immunostimulatory sequences (ISSs) of said one or more expression cassette(s) of a DNA sequence of interest are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell, while still coding for the pro- tein of interest. In one embodiment, the expression vector according to the present invention comprises one or more expression cassette(s) of a DNA sequence of interest, which code(s) for one or more non-immunogenic intrinsic protein(s), and/or which code(s) for one or more protein(s), wherein at least 50% of all immunostimulatory sequences (ISSs) are removed and/or modified, so that they do not activate an innate immune response in a mammalian cell, while still coding for the protein of interest.

Typically, this can be achieved by removing and/or modifying, in such a way that they do not activate an innate immune response in a mammalian cell, at least 10% of all human ISSs as shown in SEQ.ID.NO.: 16 and/or at least 10% of all mouse ISSs as shown in SEQ.ID.NO.: 17, in said expression cassette(s) of a DNA sequence of interest.

The one or more expression cassette(s) of a DNA sequence of interest are typically altered and/or modified so that they do not activate an innate immune response in any mammalian cell, while still coding for the protein of interest.

The present invention relates to a non-integrating expression vector with reduced ability to activate an innate immune response in a mammalian cell. Immunostimulatory sequences (ISSs)

One aspect of genetic immunization that has recently received attention is the immunostimulatory activity of DNA itself. It has e.g. been observed that DNA from bacteria can induce a non-specific immune response, possibly at least in part due to un- methylated CpG nucleotide duplexes found in the bacterial genome.

The unmethylated CpG dinucleotides are present at a higher frequency in bacterial DNA or vector DNA compared to its frequency in mammalian DNA. The CpG dinucleotides contain cytosine triphosphate deoxynucelotide ( ^" C ^" ) followed by a guanine triphosphate deoxynucleotide f ^' G ^" ). The deoxynucleotides are linked together by a phosphodiester bond ( ^" p ^" ). The unmethylated CpG sequences act as immunostimu- lants and are recognized by the pattern recognition receptor (PPR); Toll-Like Receptor 9. A receptor which is constitutively expressed on B cells and plasmacytoid dendritic cells in humans and other higher primates A further aspect, is the contribution to the immunostimulatory activity by surrounding sequences to bacterial unmethylated CpG dinucleotides. For example, the sequence GACGTT sequence is especially immunostimulatory when located in proximity to CpG dinucleotides in mouse cells and human immune cells.

The immune response elicited by immunostimulatory bacterial plasmid DNA is potent and includes secretion of several proinflammatory cytokines. In humans, the response may include fever, myalgia, and reduction in pulmonary function in cystic fibrosis. A non-integrating expression vector of the present invention comprises (a) a DNA sequence encoding a nuclear-anchoring protein operatively linked to a heterologous promoter, said nuclear-anchoring protein comprising (i) a DNA binding do main which binds to a specific DNA sequence, and (ii) a functional domain that binds to a nuclear component, or a functional equivalent thereof; and (b) a multimerized DNA sequence forming a binding site for the nuclear anchoring protein, wherein said vector lacks an origin of replication functional in any mammalian cell, and wherein at least 50% of all immunostimulatory sequences (ISSs) from the plasmid backbone and from the promoter are removed and/or modified in such a way that they do not activate an innate immune response in a mammalian cell.

Nuclear-anchoring protein

The term "nuclear-anchoring protein" as used in the present invention refers to a protein, which binds to a specific DNA sequence and is capable of providing a nuclear compartmentalization function to the vector, i.e., to a protein, which is capable of an- choring or attaching the vector to a specific nuclear compartment. In certain embodiments of the invention, the nuclear-anchoring protein is a natural protein. (Examples of such nuclear compartments are the mitotic chromatin or mitotic chromosomes, the nuclear matrix, nuclear domains like ND10 and POD etc. Examples of nuclear- anchoring proteins are the Bovine Papilloma Virus type 1 (BPV1) E2 protein, EBNA1 (Epstein-BarrVirus Nuclear Antigen 1), and High Mobility Group (HMG) proteins etc.

The term "functional equivalent of a nuclear-anchoring protein" as used in the present invention refers to a protein or a polypeptide of natural or non-natural origin having the properties of the nuclear-anchoring protein. ln certain other embodiments of the invention, the nuclear-anchoring protein of the invention is a recombinant protein. In certain specific embodiments of the invention, the nuclear-anchoring protein is a fusion protein, a chimeric protein, or a protein obtained by molecular modeling. A fusion protein, or a protein obtained by molecular modeling in connection with the present invention is characterized by its ability to bind to a nuclear component and by its ability to bind sequence-specifically to DNA. In a preferred embodiment of the invention, such a fusion protein is encoded by a vector of the invention which also contains the specific DNA sequence to which the fu- sion/chimeric protein binds. Nuclear components include, but are not limited to chro- matin, the nuclear matrix, the ND10 domain and POD. In order to reduce the risk of interference with the expression of genes endogenous to the host cell, the DNA binding domain and the corresponding DNA sequence is preferably non-endogenous to the host cell/host organism. Such domains include, but are not limited to, the DNA binding domain of the Bovine Papilloma Virus type 1 (BPV1) E2 protein , Epstein-Barr Virus Nuclear Antigen 1 (EBNA1), and High Mobility Group (HMG) proteins (HMG box).

The vector of the invention can further comprise a "DNA sequence of interest", that encodes a protein (including a peptide or polypeptide), e.g., that is a therapeutic. In certain embodiments of the invention, the DNA sequence of interest encodes a biologically active RNA molecule, such as an antisense RNA molecule or a ribozyme.

The expression vectors of the invention carrying an expression cassette for a gene of a nuclear-anchoring protein and multimerized binding sites for said nuclear-anchoring protein spread in a proliferating host cell population. This means that a high copy- number of vectors or plasmids are delivered into the target cells and the use of the segregation/partitioning function of the nuclear-anchoring protein and its multimerized binding sites assures the distribution of the vector to the daughter cells during cell division.

The vector of the invention lacks a papilloma virus origin of replication. Further, in a preferred embodiment, the vector of the invention lacks an origin of replication functional in a/any mammalian cell. The omission of a papilloma virus origin of replication or a mammalian origin of replication constitutes an improvement over prior art vectors for several reasons. (1 ) Omission of the origin of replication reduces the size of the vector of the invention compared to prior art vectors. Such a reduction in size increases the stability of the vector and facilitates uptake by the host cell. (2) Omission of the origin of replication reduces the risk for recombination with the host cell's genome, thereby reducing the risk of unwanted side effects. (3) The omission of the origin of replication allows control of the dosage simply by adjusting the amount of vector administered. In contrast, with a functioning origin of replication, replication of the vector has to be taken into consideration when determining the required dosage. (4) If the vector is not administered to a host organism continually, the lack of an origin of replication allows the host organism to clear itself of the vector, thus providing more control over the levels of DNA sequences to be expressed in the host organism. Further, the ability of the organism to clear itself of the vector will be advantageous if the presence of the vector is required only during the course of a therapy but is undesirable in a healthy individual. The gene of a nuclear-anchoring protein useful in the vectors of the present invention can be any suitable DNA sequence encoding a natural or artificial protein, such as a recombinant protein, a fusion protein or a protein obtained by molecular modeling techniques, having the required properties. Thus the gene of a natural nuclear- anchoring protein, which contains a DNA binding domain capable of binding to a spe- cific DNA sequence and a functional domain capable of binding to a nuclear component, can be that of a viral protein, such as the E2 protein of Bovine Papilloma Virus or the EBNA1 (Epstein-Barr Virus Nuclear Antigen 1) of the Epstein-Barr Virus, a eu- karyotic protein such a one of the High Mobility Group (HMG) proteins or a like protein, or a prokaryotic protein. Alternatively, the gene of a nuclear-anchoring protein, which contains a DNA binding domain capable of binding to a specific DNA sequence and a functional domain capable of binding to a nuclear component, can also be comprised of DNA sequences, which encode a domain from a cellular protein having the ability to attach to a suitable nuclear structure, such as to mitotic chromosomes, the nuclear matrix or nuclear domains like ND10 or POD.

Alternatively, the DNA sequence, which encodes a non-natural or artificial protein, such as a recombinant protein or a fusion protein or a protein obtained by molecular modeling, which contains a DNA binding domain capable of binding to a specific DNA sequence of, e.g., a papilloma virus, such as the DNA binding domain of the E2 pro- tein of the BPV1 , but in which the N-terminus of the nuclear-anchoring protein, e.g. that of the E2 protein, has been replaced with domains of any suitable protein of similar capacity, for example, with the N-terminal domain of Epstein-Barr Virus Nuclear Antigen 1 sequence, can be used. Similarly, DNA sequences, which encode a recombinant protein or a fusion protein, which contains a functional domain capable of bind- ing to a nuclear component, e.g., the N-terminal functional domain of a papilloma virus, such as the E2 protein of the BPV1 , but in which the C-terminal DNA-binding di- merization domain of the nuclear-anchoring protein, e.g., that of the E2 protein, has been replaced with domains of any protein of a sufficient DNA-binding strength, e.g., the DNA binding domain of the BPV-1 E2 protein and the EBNA-1 , can be used.

In a preferred embodiment of the invention, the nuclear-anchoring protein is a chro- matin-anchoring protein, which contains a DNA binding domain, which binds to a specific DNA sequence, and a functional domain capable of binding to mitotic chromatin. A preferred example of such a chromatin-anchoring protein and its multimerized bind- ing sites useful in the present invention are the E2 protein of Bovine Papilloma Virus type 1 and E2 protein multimerized binding sites. In the case of E2, the mechanism of the spreading function is due to the dual function of the E2 protein: the capacity of the E2 protein to attach to mitotic chromosomes through the N-terminal domain of the protein and the sequence-specific binding capacity of the C-terminal domain of the E2 protein, which assures the tethering of vectors, which contain a multimerized E2 binding site, to mitotic chromosomes. A segregation/partitioning function is thus provided to the vectors.

In another preferred embodiment of the invention, the expression cassette of a gene of the chromatin-anchoring protein comprises a gene of any suitable protein of cellular, viral or recombinant origin having analogous properties to E2 of the BPV1 , i.e., the ability to attach to the mitotic chromatin through one domain and to cooperatively bind DNA through another domain to multimerized binding sites specific for this DNA binding domain.

In a specific embodiment, sequences obtained from BPV1 , are used in the vectors of the present invention, they are extensively shortened in size to include just two elements from BPV1. First, they include the E2 protein coding sequence transcribed from a heterologous eukaryotic promoter and polyadenylated at the heterologous pol- yadenylation site. Second, they include E2 protein multiple binding sites incorporated into the vector as a cluster, where the sites can be as head-to-tail structures or can be included into the vector by spaced positioning. Both of these elements are necessary and, surprisingly, sufficient for the function of the vectors to spread in proliferating cells. Similarly, when DNA sequences based of other suitable sources are used in the vectors of the present invention, the same principles are applied.

According to the present invention, the expression cassette of a gene of a nuclear- anchoring protein, which contains a DNA binding domain capable of binding to a specific DNA sequence and a functional domain capable of binding to a nuclear compo- nent, such as an expression cassette of a gene of a chromatin-anchoring protein, like BPV1 E2, comprises a heterologous eukaryotic promoter, the nuclear-anchoring protein coding sequence, such as a chromatin-anchoring protein coding sequence, for instance the BPV1 E2 protein coding sequence, and a poly A site. Different heterologous, eukaryotic promoters, which control the expression of the nuclear-anchoring protein, can be used. Nucleotide sequences of such heterologous, eukaryotic promoters are well known in the art and are readily available. Such heterologous eukaryotic promoters are of different strength and tissue-specificity. In a preferred embodiment, the nuclear anchoring protein is expressed at low levels. The multimerized DNA binding sequences, i.e., DNA sequences containing multimeric binding sites, as defined in the context of the present invention, are the region, to which the DNA binding dimerization domain binds. The multimerized DNA binding sequences of the vectors of the present invention can contain any suitable DNA binding site, provided that it fulfills the above requirements.

In a preferred embodiment, the multimerized DNA binding sequence of a vector of the present invention can contain any one of known 17 different affinity E2 binding sites as a hexamer or a higher oligomer, as a octamer or a higher oligomer, as a decamer or higher oligomer. Oligomers containing different E2 binding sites are also applica- ble. Specifically preferred E2 binding sites useful in the vectors of the present invention are the BPV1 high affinity sites 9 and 10, affinity site 9 being most preferred. When a higher oligomer is concerned, its size is limited only by the construction circumstances and it may contain from 6 to 30 identical binding sites. Preferred vectors of the invention contain 10 BPV-1 E2 binding sites 9 in tandem. When the multimer- ized DNA binding sequences are comprised of different E2 binding sites, their size and composition is limited only by the method of construction practice. Thus they may contain two or more different E2 binding sites attached to a series of 6 to 30, most preferably 10, E2 binding sites. The Bovine Papilloma Virus type 1 genome contains 17 E2 protein binding sites which differ in their affinity to E2. The E2 binding sites are described in Li et al. (Genes Dev 3(4) (1989) 510-526), which is incorporated by reference in its entirety herein.

Alternatively, the multimerized DNA binding sequences may be composed of any suitable multimeric specific sequences capable of inducing the cooperative binding of the protein to the plasmid, such as those of the EBIMA1 or a suitable HMG protein. 21x30bp repeats of binding sites for EBNA-1 are localized in the region spanning from nucleotide position 7421 to nucleotide position 8042 of the Epstein-Barr virus genome. These EBNA-1 binding sites are described in the following references: Rawlins et al., Cell 42(3) (1985) 859-868; Reisman et al., Mol Cell Biol 5(8) (1985) 1822-1832; and Lupton and Levine, Mol Cell Biol 5(10) (1985) 2533-2542, all three of which are incorporated by reference in their entireties herein.

The position of the multimerized DNA binding sequences relative to the expression cassette for the DNA binding dimerization domain is not critical and can be any position in the plasmid. Thus the multimerized DNA binding sequences can be positioned either downstream or upstream relative to the expression cassette for the gene of interest, a position close to the promoter of the gene of interest being preferred. The vectors of the invention also contain, where appropriate, a suitable promoter for the transcription of the gene or genes or the DNA sequences of interest, additional regulatory sequences, polyadenylation sequences and introns. Preferably the vectors may also include a bacterial plasmid origin of replication and one or more genes for selectable markers to facilitate the preparation of the vector in a bacterial host and a suitable promoter for the expression the gene for antibiotic selection.

The selectable marker can be any suitable marker allowable in DNA vaccines, such a kanamycin or neomycin, and others. In addition, other positive and negative selection markers can be included in the vectors of the invention, where applicable. The vectors of the present invention only comprise the DNA sequences, for instance BPV1 DNA sequences, which are necessary and sufficient for long-term maintenance. All superfluous sequences, which may induce adverse reactions, such as oncogenic sequences, have been deleted. Thus in preferred vectors of the invention the E2 cod- ing sequence is modified by mutational analysis so that this expresses only E2 protein and overlapping E3, E4 and E5 sequences have been inactivated by the introduction of mutations, which inactivate the translation from Open Reading Frames for E3, E4 and E5. The vector of the invention does not contain a papilloma virus origin of replication. Preferably, the vector of the invention further does not contain an origin of replication functional in a/any mammalian cell or a/any mammal.

The vectors of the present invention in certain embodiments correspond to and/or consist of and/or encompass and/or comprise a nucleic acid sequence selected from any one of the nucleic acid sequences as shown or represented as elements of a plasmid as shown in any one of figures 11 , 12, 15, 16, 17, 18, 19, 21 , 22, 23, 24, and 42.

A non-integrating expression vector according to the present invention is e.g. as shown in SEQ.ID.Nos:1-21.

Furthermore, the vectors of the present invention are not host specific, since the expression of the nuclear-anchoring protein, such as the E2 protein, is controlled by non-native or heterologous promoters. Depending on the particular promoter chosen, these promoters may be functional in a broad range of mammalian cells or they can be cell or tissue specific. Examples of promoters for the nuclear-anchoring protein, such as for the E2 protein, useful in the vectors of the present invention are thymidine kinase promoters, Human Cytomegalovirus Immediate Early Promoter, Rous Sarcoma Virus LTR and like. For the expression of the gene of interest, preferred promoters are strong promoters assuring high levels of expression of the gene of interest, an example for such a promoter is the Human Cytomegalovirus Immediate Early Promoter. THE VECTORS OF THE INVENTION AS VEHICLES FOR EXPRESSION OF A DNA SEQUENCE OF INTEREST

A gene, genes or a DNA sequence or DNA sequences to be expressed via a vector of the invention can be any DNA sequence of interest, whose expression is desired.

Particularly, the vectors of the invention may contain genes or DNA sequences for inherited or acquired genetic defects, such as sequences of differentiation antigens for melanoma, like a Tyrosinase A coding sequence or a coding sequence of beta- catenins.

In another preferred embodiment of the invention, the vectors contain a gene encoding proteins relating to cancer or other mutational diseases, preferably diseases related to immune maturation and regulation of immune response towards self and non- self, such as the APECED gene.

In yet another preferred embodiment of the invention, the vectors contain any DNA sequence coding for a protein that is defective in any hereditary and/or single gene hereditary disease. Known gene targets for hereditary diseases are in the present context e.g. selected from the non-exclusive list consisting of ABCA4 Retinitis Pigmentosa, ABCC9 Dilated Cardiomyopathy 10, ABCD1 X-Linked Adrenoleukodystrophy, ACADVL Very Long Chain Acyl-Coenzyme A Dehydrogenase Deficiency, ACTA2 Thoracic Aortic Aneurysms and Aortic Dissections, ACTC1 Familial Hypertrophic Cardiomyopathy, ACTN2 Dilated Cardiomyopathy 1AA, ADA Severe Combined Immunodeficiency, AIPL1 Leber Congenital Amaurosis, AIRE Autoimmune Polyendocrine Syndrome, AKAP9 Long QT Syndrome, Autosomal Dominant, AKR1 B1 Androgen Insensitivity Syndrome, ALPL Hypophosphatasia, AMT Glycine Encephalopathy, ANK2 Long/Short QT Syndrome, Autosomal Dominant, APC APC-Associated Polyposis Conditions, APP Early- Onset Familial Alzheimer Disease, APTX Ataxia with Oculomotor Apraxia Type 2, ARL6, Retinitis Pigmentosa, ARSA Arylsulfatase A Deficiency, ASL Argininosuccinate Lyase Deficiency, ASPA Canavan, ATL1 Spastic Paraplegia-3A, ATM Ataxia- Telangiectasia, ATP2A2 Darier Disease, ATP7A Menkes/ATP7A-Related Copper Transport Disease, ATP7B Wilson Disease, ATXN1 Spinocerebellar Ataxia 1 , ATXN2 Spinocerebellar Ataxia 2, ATXN7 Spinocerebellar Ataxia 7, BAG3 Dilated Cardiomyo- pathy 1HH, BCKDHA Maple Syrup Urine Disease, BMPR1A, Juvenile Polyposis Syndrome, BTD Biotinidase Deficiency, BTK Agammaglobulinemia, X-Linked, Type 1 , CA4 Retinitis Pigmentosa, CACNA1C Brugada Syndrome, CACNB2 Brugada Syndrome, CALR3 Familial Hypertrophic Cardiomyopathy, CAPN3 Limb-Girdle Muscular Dystrophy Type 2A - Calpainopathy, CASQ2 Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), CAV3 Familial Hypertrophic Cardiomyopathy, CCDC39 Primary Ciliary Dyskinesia, CCDC40 Primary Ciliary Dyskinesia, CDH23 Usher Syndrome Type 1 , CEP290 Leber Congenital Amaurosis, CERKL Retinitis Pigmentosa, CFTR Cystic Fibrosis, CHAT Congenital Myasthenic Syndromes, CHD7 Charge Syn- drome, CHEK2 Li-Fraumeni Syndrome, CHM Choroideremia, CHRNA1 Congenital Myasthenic Syndromes, CHRNB1 Congenital Myasthenic Syndromes, CHRND Congenital Myasthenic Syndromes, CHRNE Congenital Myasthenic Syndromes, CLCN1 Myotonia Congenita, CNGB1 Retinitis Pigmentosa, COL11A1 Stickler Syndrome, AD, COL11A2 Inherited Deafness, COL1A1 Osteogenesis Imperfecta, COL1A2, Osteo- genesis Imperfecta, COL2A1 Stickler Syndrome, AD, COL3A1 Ehlers-Danlos Syndrome, COL4A1 Thoracic Aortic Aneurysms and Aortic Dissections, COL4A5 Alport Syndrom, BCKDHB Maple Syrup Urine Disease, BEST1 Retinitis Pigmentosa, COL5A1 Ehlers-Danlos Syndrome, Classic Type, COL5A2 Ehlers-Danlos Syndrome, Classic Type, COL7A1 Epidermolysis Bullosa Simplex, COL9A1 Stickler Syndrome, CRB1 Leber Congenital Amaurosis, CRX Retinitis Pigmentosa, CTDP1 , Congenital Cataracts, Facial Dysmorphism, and Neuropathy, CTNS Cystinosis, CYP27A1 Cere- brotendinous Xanthomatosis, DBT Maple Syrup Urine Disease, DCX Double Cortex Syndrome, DES Dilated Cardiomyopathy, DHCR7 Smith-Lemli-Opitz Syndrome, DKC1 , Dyskeratosis Congenita DLD, Maple Syrup Urine Disease DMD, Du- chenne/Becker Muscular Dystrophy, DIMAH5 Primary Ciliary Dyskinesia, DIMAH9 Primary Ciliary Dyskinesia, DNAH11 Primary Ciliary Dyskinesia, DNAI1 , Primary Ciliary Dyskinesia, DNAI2 Primary Ciliary Dyskinesia, DNM2 Charcot-Marie-Tooth Disease Type 2B, DOK7 Congenital Myasthenic Syndromes, DSC2 Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy, DSG2 Arrhythmogenic Right Ventricular Dys- plasia/Cardiomyopathy, DSP Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy, DYSF Dysferiinopathy, ELN Supravalvular Aortic Stenosis, EMD Emery-Dreifuss Muscular Dystrophy, X-Linked, ENG Hereditary Hemorrhagic Telangiectasia, EXT1 Exostoses, Multiple, Type 1, EYA1 Branchiootorenal Spectrum Disorders, EYS Retinitis Pigmentosa, F8 Hemophilia A, F9 Hemophilia B, FANCA Fan- coni Anemia, FANCC Fanconi Anemia, FANCF Fanconi Anemia, FANCG Fanconi Anemia, FBN1 Marfan Syndrome, FBX07 Parkinson Disease, FGFR1 FGFR-Related Craniosynostosis Syndromes, FGFR3 Hypochondroplasia, FM03 Trimethylaminuria, FOXL2 Blepharophimosis-Ptosis-Epicanthus Inversus, FRG1 Facioscapulohumeral Muscular Dystrophy, FRMD7 FRMD7-Related Infantile Nystagmus, FSCN2 Retinitis Pigmentosa, FXN Friedreich Ataxia, GAA Pompe Disease -GSD II, GALT Galactosemia, GATA4 Atrial Septal Defect, GBA Gaucher Disease, GBE1 Glycogen Storage Disease Type VI, GCSH Glycine Encephalopathy, GDF5 Brachydactyly, GJB2 Inherited Deafness, Top Genes, GJB3 Inherited Deafness, Top Genes, GJB6 Inherited Deafness, Top Genes, GLA Fabry Disease, GLDC Glycine Encephalopathy, GNE In- elusion Body Myopathy 2, GNPTAB Mucolipidosis II, GPC3 Wilms Tumor, Classical, GPD1 L Brugada Syndrome, GPR143 Ocular Albinism, X-Linked, GUCY2D Leber Congenital Amaurosis, HBA2 Alpha-Thalassemia - Southeast Asia, HBB Sickle Cell Disease Beta-Thalassemia, HCN4 Brugada Syndrome, HEXA Hexosaminidase A Deficiency, HFE HFE-Associated Hereditary Hemochromatosis, HIBCH Beta- Hydroxyisobutyryl CoA Deacylase Deficiency (HIBCH Deficiency), HMBS Hy- droxymethylbilane Synthase (HMBS) Deficiency, HR Alopecia Universalis Congenita (ALUNC), IDS Hunter Syndrome (MPSII), IDUA Hurler Syndrome (MPSI), IKBKAP Familial Dysautonomia (HSAN III), IL2RG X-Linked SCIDS, IMPDH1 Leber Congenital Amaurosis, ITGB4 Epidermolysis Bullosa Simplex, JAG1 Alagille Syndrome, JUP Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy, KCNE1 Long QT Syndrome, Autosomal Dominant, KCNE2 Long QT Syndrome, Autosomal Dominant, KCNE3 Brugada Syndrome, KCNH2 Long QT Syndrome, Autosomal Dominant, KCNJ2 Short QT Syndrome, KCNQ1 Long QT Syndrome, Autosomal Dominant, KCNQ4 Inherited Deafness, KIAA0196 Spastic Paraplegia 8, KLHL7 Retinitis Pigmen- tosa, KRAS Noonan Syndrome, KRT5 Epidermolysis Bullosa Simplex, KRT14 Epidermolysis Bullosa Simplex, L1CAM Spastic Paraplegia Type 1 - L1 Syndrome, LAMB3 Epidermolysis Bullosa Simplex, LAMP2 Dilated Cardiomyopathy, LDB3 Dilated Cardiomyopathy, LMNA Limb-Girdle Muscular Dystrophy, Type 1B, LRAT Retinitis Pigmentosa, LRRK2 Parkinson Disease, MAPRE2 Retinitis Pigmentosa, MAPT Par- kinson-Dementia Syndrome, MC1 R Oculocutaneous Albinism Type 2, MECP2 MECP2-Rett Syndrome, MED12 Fryns Syndrome, MEN1 Multiple Endocrine Neoplasia Type 1 , MERTK Retinitis Pigmentosa, MFN2 Charcot-Marie-Tooth Neuropathy Type 2A, MLH1 Turcot Syndrome, MMAA Methylmalonic Acidemia, MMAB Methylmalonic Acidemia , MMACHC Methylmalonic Acidemia, MPZ Charcot-Marie-Tooth Neu- ropathy Type 1B, MSH2 Turcot Syndrome, MTM1 X-Linked Myotubular Myopathy, MUT Methylmalonic Acidemia, MYBPC3 Familial Hypertrophic Cardiomyopathy, MYH11 Thoracic Aortic Aneurysms and Aortic Dissections, MYH6 Familial Hypertrophic Cardiomyopathy, MYH7 Familial Hypertrophic Cardiomyopathy, MYL2 Familial Hypertrophic Cardiomyopathy, MYL3 Familial Hypertrophic Cardiomyopathy, MYLK Familial Hypertrophic Cardiomyopathy, MY07A Usher Syndrome Type 1 , MYOZ2 Familial Hypertrophic Cardiomyopathy, NF1 Neurofibromatosis Type 1 , NF2 Neurofibromatosis Type 2, NIPBL Cornelia de Lange Syndrome, NKX2-5 Tetralogy of Fallot, NPC1 Niemann-Pick Disease Type C1 , NPC2 Niemann-Pick Disease Type C2, NR2E3 Retinitis Pigmentosa , NRAS Noonan Syndrome, NSD1 Sotos Syndrome, NUDT19 Retinitis Pigmentosa, OCA2 Oculocutaneous Albinism Type 2, OCRL Lowe Syndrome, OTC Ornithine Transcarbamylase Deficiency, PABPN1 Oculopharyngeal Muscular Dystrophy, PAFAH1B1 Lissencephaly 1 , PAH Phenylketonuria (PKU), PAX3 Waardenburg Syndrome, Type 1 , PAX6 Aniridia, PCDH15 Usher Syndrome Type 1 , PEX1 Zellweger Syndrome, ΡΕΞΧ3 Peroxisome Biogenesis, Zellweger, ΡΕΞΧ5 Neonatal Adrenoleucodystrophy, ΡΕΞΧ10 Peroxisome Biogenesis, Zellweger, PEX13 Peroxisome Biogenesis, Zellweger, ΡΕΞΧ14 Peroxisome Biogenesis, Zellweger, ΡΕΞΧ19 Peroxisome Biogenesis, Zellweger, PEX26 Peroxisome Biogenesis, Zellweger, PINK1 Parkinson Disease, PKD1 Polycystic Kidney Disease, Autosomal Dominant, PKD2 Polycystic Kidney Disease, Autosomal Recessive, PKHD1 Polycystic Kidney Disease, Autosomal Recessive, PKP2 Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy, PLEC Epidermolysis Bullosa Simplex, PLN Dilated Cardiomyopathy 1 P, PLOD1 Ehlers-Danlos Syndrome, Kyphoscoliotic Form, PMM2 Congenital Disorder of Glycosylation Type 1a , PMP22 Charcot-Marie-Tooth Neuropathy Type 1A, POLG Alpers Syndrome , PPT1 Ceroid Lipofuscinoses (Batten Disease), PRCD Retinitis Pigmentosa, PRKAG2 Familial Hypertrophic Cardiomyopathy, PROM1 Retinitis Pigmentosa, PRPF8 Retinitis Pigmentosa , PRPF31 Retinitis Pigmentosa, PRPH2 Retinitis Pigmentosa, PSEN1 Early-Onset Familial Alzheimer Disease, PSEN2 Early-Onset Familial Alzheimer Disease, PTCH1 Holoprosencephaly-7 & Basal Cell Nevus Syndrome, PTPN11 Noonan Syndrome, RAF1 Noonan Syndrome, RAG1 Severe Combined Immunodeficiency, RAG2 Severe Combined Immunodeficiency, RAM Smith-Magenis Syndrome, APSN Congenital Myasthenic Syndromes, RB1 Retinoblastoma, RDH12 Leber Congenital Amaurosis, RET Multiple Endocrine Neoplasia Type 2, RHO Retinitis Pigmentosa, ROR2 Brachydactyly, Type B1 , RP9 Retinitis Pigmentosa , RPE65 Leber Congenital Amaurosis, RPGR Retinitis Pigmen- tosa, RPGRIP1 Leber Congenital Amaurosis, RPL11 Diamond-Blackfan Anemia, RPL35A Diamond-Blackfan Anemia, RPS6KA3 Coffin-Lowry Syndrome, RPS7 Familial Hypertrophic Cardiomyopathy, RPS10 Diamond-Blackfan Anemia, RPS19 Diamond-Blackfan Anemia, RPS24 Diamond-Blackfan Anemia, RPS26 Diamond- Blackfan Anemia, RS1 X-Linked Juvenile Retinoschisis, RSPH4A Primary Ciliary Dyskinesia, RSPH9 Primary Ciliary Dyskinesia, RYR1 Malignant Hyperthermia Susceptibility, RYR2 Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy, SALL4 Duane Syndrome - Autosomal Dominant, SCN1 B Brugada Syndrome, SCN3B Bru- gada Syndrome, SCN4B Long QT Syndrome, Autosomal Dominant, SCN5A Brugada Syndrome, SCN9A SCN9A-Related Inherited Erythromelalgia, SEMA4A Retinitis Pigmentosa, SERPINA1 Alpha-1 -Antitrypsin Deficiency, SERPING1 Angioedema, Hereditary, Types I and II, SGCD Dilated Cardiomyopathy, SH3BP2 Cherubism, SIX1 Branchiootorenal Spectrum Disorders, SIX5 Branchiootorenal Spectrum Disorders, SLC25A4 Familial Hypertrophic Cardiomyopathy, SLC25A13 Citrin Deficienc,

SLC26A4 Pendred Syndrome/Syndromic Deafness, SMAD3 Thoracic Aortic Aneu- rysms and Aortic Dissections, SMAD4 Juvenile Polyposis Syndrome, SNCA Parkinson Disease, SNRNP200 Retinitis Pigmentosa, SNTA1 Long QT Syndrome, Autosomal Dominant, SOD1 Amyotrophic Lateral Sclerosis (Lou Gehrig's Disease), SOS1 Noonan Syndrome, SOX9 Campomelic Dysplasia, SPATA7 Retinitis Pigmentosa, SPG7 Spastic Paraplegia 7, STARD3 Cardiomyopathy (Dilated), TAF1 X-Linked Dys- tonia-Parkinsonism, TAZ Cardiomyopathy (Dilated), TBX5 Holt-Oram Syndrome, TCOF1 Treacher Collins Syndrome, TGFBR1 Thoracic Aortic Aneurysms and Aortic Dissections, TGFBR2 Thoracic Aortic Aneurysms and Aortic Dissections, TMEM43 Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy, TNNC1 Dilated Cardiomyopathy, TNNI3 Dilated Cardiomyopathy, TNNT1 Nemaline Myopathy, TNNT2 Familial Hypertrophic Cardiomyopathy, TNXB Ehlers-Danlos Syndrome, Hypermobili- ty Type, TOPORS Retinitis Pigmentosa, TP53 Li-Fraumeni Syndrome, TPM1 Familial Hypertrophic Cardiomyopathy, TSC1 Tuberous Sclerosis Complex, TSC2 Tuberous Sclerosis Complex, TTPA Ataxia with Vitamin E Deficiency, TTR Familial Transthyretin Amyloidosis, TULP1 Retinitis Pigmentosa, TWIST1 Saethre-Chotzen Syndrome, TXNDC3 Primary Ciliary Dyskinesia, TYR Oculocutaneous Albinism Type 1 , USH1C Usher Syndrome Type 1 , USH2A Usher Syndrome Type 2, VCL Familial Hypertrophic Cardiomyopathy, VHL von Hippel-Lindau Syndrome, WAS Wiskott-Aldrich Syndrome, WRN Werner Syndrome, WT1 Wilms Tumor, Classical. ln another preferred embodiment of the invention, the vectors contain any DNA sequence coding for at least one anti-tumor substance, a protein and/or macromolecular drug to be delivered and produced in vivo. Typically, such an anti-tumor substance, protein and/or macromolecular drug is in selected from the non-exclusive list consist- ing of antimetabolites including anti-folates, fluoropyrimidines, deoxynucleoside analogues and thiopurines, such as deoxcytidine, gemcitabine, decitabine, methotrexate, pemetrexed, fluorouracil, capecitabine cytarabine, gemcitabine, decitabine, vidaza, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine and mercap- topurine, anti-microtubule agents including vinca alkaloids and taxanes, such as vin- cristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, podophyl- lotoxin .etoposide and teniposide, topoisomerase inhibitors including irinotecan, topo- tecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin, and cytotoxic antibiotics including anthracyclines, actinomycin, bleomycin, plicamycin, mitomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, mitoxantrone, actinomycin, bleomycin, and mitomycin.

In a presently prefered embodiment, a non-integrating expression vector according to the invention comprises one or more expression cassette(s) of a DNA sequence encoding at least one of

a. a heavy chain and

b. a light chain, of

a tumour specific antibody. Ttypically, said tumour specific antibody is an angiogene- sis inhibitor, e.g. selected from one of: A.4.6.1 and Bevacizumab. Alternatively, a non-integrating expression vector according to the invention comprises one or more expression cassette(s) of a DNA sequence encoding at least one of a. a heavy chain and

b. a light chain, of a virus specific antibody.

It has surprisingly been shown that administration of a non-integrating expression vector according to the invention which comprises one or more expression cassette(s) of a DNA sequence encoding both a heavy chain and a light chain, of a monoclonal an- tibody gives rise to a rapid high MAb level (see example 5) in blood in all animals treated.

This can be especially advantageous in the treatment of conditions whith a rapid dis- ease progression, such as in viral infections or cancer treatment, wherein time to treatment is crucial. Thus the present invention in one presently preferred embodiment relates to a non-integrating expression vector according to the invention which comprises one or more expression cassette(s) of a DNA sequence encoding both a heavy chain and a light chain for use in treatment of a viral infection and/or cancer.

The method of the invention for the preparation of the vectors of the invention comprises the following steps: (A) cultivating a host cell containing a vector of the invention, and (B) recovering the vector. In certain specific embodiments, step (A) is preceded by transforming a host cell with a vector of the invention.

The vectors of the invention are preferably amplified in a suitable bacterial host cell, such as Escherichia coli. The vectors of the invention are stable and replicate at high copy numbers in bacterial cells. If a vector of the invention is to be amplified in a bacterial host cell, the vector of the invention contains a bacterial origin of replication. Nucleotide sequences of bacterial origins of replication are well known to the skilled artisan and can readily be obtained.

Upon transfection into a mammalian host in high copy number, the vector spreads along with cell divisions and the number of cells carrying the vector increases without the replication of the vector, each cell being capable of expressing the protein of interest.

The vectors of the invention result in high expression of the desired protein or macro- molecular drug.

The present invention further relates to pharmaceutical compositions comprising the expression vectors of the present invention or a mixture of said vectors in a suitable pharmaceutical carrier. Said pharmaceutical compositions are formulated using standard methods for administration by any conventional route of administration, i.e. intramuscularily, intradermally, subcutan, intravenous, intranasal, systemically, topically, local, etc.

The vectors of the present invention carrying the mechanism of spreading in the host cell find numerous applications in gene therapy, in gene transfer and as therapeutic agents. The vectors of the invention can be used to deliver a normal gene to a host having a gene defect, thus leading to a cure or therapy of a genetic disease. Furthermore, the vectors of the invention can deliver suitable genes of marker substances to nucleus, to be used in studies of cellular function or in diagnostics. Finally, the vectors of the invention can be used to specifically deliver a gene of macromolecular drug to the nucleus, thus enabling the development of novel therapeutic principles to treat and cure diseases, where the expression of the drug in the site of action, the cell nucleus, is of importance. These drugs can be chemical macromolecules, such as any proteins or polypeptides with therapeutic or curative effect, which interfere with any of the nuclear mechanisms, such as the replication or transcription or the transport of substances to and from the nucleus.

The vectors of the present invention are also useful in complementing malfunctioning of the brain due to the loss of specific dopamine-ergic neurons leading to the irre- versible neurodegeneration, which is cause for Parkinson's disease, by expressing genes involved into synthesis of dopamine, like tyrosine hydroxylase, as well as other genes deficiency of which would have the similar effect. The vectors of the present invention are also useful for the expression of proteins and peptides regulating the brain activity, like dopamine receptors, CCK-A and CCK-B receptors, as well as neu- rotrophic factors, like GDNF, BDNF and other proteins regulating the brain activity. Further, the vectors of the present invention are useful for a long-term expression of factor IX in hepatocytes and alfal -antitrypsin in muscle cells with the aim of complementing respective deficiencies of the organism.

TARGET DISEASES AND DISORDERS

In certain embodiments, a vector of the invention contains a DNA sequence of interest that encodes a protein or a peptide. Upon administering of such a vector to a subject, the protein or peptide encoded by the DNA sequence of interest is expressed. ln specific embodiments, the vector of the invention is used to treat and/or prevent an infectious disease and/or a condition caused by an infectious agent. Such diseases and conditions include, but are not limited to, infectious diseases caused by bacteria, viruses, fungi, protozoa, helminths, and the like. In a more specific embodiment of the invention, the infectious disease is Acquired Immunodeficiency Syndrome (AIDS). Therefore, one embodiment relates to the use of the vector of the invention for the expression of neutralizing antibodies, in particular for the expression of neutralizing antibodies against AIDS and/or for editing T-cells in patients' immune systems to become resistant to the HIV virus.

In other specific embodiments, the vector of the invention is used to treat and/or prevent a neoplastic disease in a subject. In these embodiments, the DNA sequence of interest encodes a protein or peptide that is specific to or associated with the neoplastic disease. By way of non-limiting example, the neoplastic disease can be a fi- brosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendo- theliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glio- ma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobu- linemia, and heavy chain disease, etc.

In certain other embodiments of the invention, the DNA sequence of interest encodes a protein that is non-functional or malfunctioning due to an inherited disorder or an acquired mutation in the gene encoding the protein. Such genetic diseases include, but are not limited to, metabolic diseases, e.g., Atherosclerosis (affected gene: AP- OE); cancer, e.g., Familial Adenomatous Polyposis Coli (affected gene: APC gene); auto-immune diseases, e.g., autoimmune polyendocrinopathy-candidosis-ectodermal dysplasia (affected gene: APECED); disorders of the muscle, e.g., Duchenne muscular dystrophyvaccines (affected gene: DMD); diseases of the nervous system, e.g., Alzheimer's Disease (affected genes: PS1 and PS2).

In even other embodiments, the vectors of the invention are used to treat and/or pre- vent diseases and disorders caused by pathologically high activity of a protein. In these embodiments of the invention, the DNA sequence of interest encodes an antagonist of the overactive protein. Such antagonists include, but are not limited to, an- tisense RNA molecules, ribozymes, antibodies, and dominant negative proteins. In specific embodiments of the invention, the DNA sequence of interest encodes an in- hibitor of an oncogene.

In certain embodiments, the DNA sequence of interest encodes a molecule that antagonizes neoplastic growth. In specific embodiments of the invention, the DNA sequence of interest encodes a tumor suppressor, such as, but not limited to, p53. In other specific embodiments, the DNA sequence of interest encodes an activator of apoptosis, such as but not limited to, a Caspase.

The invention provides methods, whereby a DNA sequence of interest is expressed in a subject. In certain embodiments, a vector containing one or more expression cas- settes of a DNA sequence of interest is administered to the subject, wherein the subject does not express the Bovine Papilloma Virus E1 protein.

THERAPEUTIC METHODS FOR USE WITH THE INVENTION RECOMBINANT DNA

In various embodiments of the invention, the vector of the invention comprises one or more expression cassettes comprising a DNA sequence of interest. The DNA sequence of interest can encode a protein and/or a biologically active RNA molecule. In either case, the DMA sequence is inserted into the vector of the invention for expression in recombinant cells or in cells of the host in the case of gene therapy.

An expression cassette, as used herein, refers to a DNA sequence of interest opera- bly linked to one or more regulatory regions or enhancer/promoter sequences which enables expression of the protein of the invention in an appropriate host cell. "Opera- bly-linked" refers to an association in which the regulatory regions and the DNA sequence to be expressed are joined and positioned in such a way as to permit transcription, and in the case of a protein, translation.

The regulatory regions necessary for transcription of the DNA sequence of interest can be provided by the vector of the invention. In a compatible host-construct system, cellular transcriptional factors, such as RNA polymerase, will bind to the regulatory regions of the vector to effect transcription of the DNA sequence of interest in the host organism. The precise nature of the regulatory regions needed for gene expression may vary from host cell to host cell. Generally, a promoter is required which is capable of binding RNA polymerase and promoting the transcription of an operably- associated DNA sequence. Such regulatory regions may include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. The non-coding region 3' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.

Both constitutive and inducible regulatory regions may be used for expression of the DNA sequence of interest. It may be desirable to use inducible promoters when the conditions optimal for growth of the host cells and the conditions for high level expression of the DNA sequence of interest are different. (Examples of useful regulatory regions are provided below. In order to attach DNA sequences with regulatory functions, such as promoters, to the DNA sequence of interest or to insert the DNA sequence of interest into the cloning site of a vector, linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of the cDNAs by techniques well known in the art. Cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation. Alter- natively, a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA by use of PCR with primers containing the desired restriction enzyme site. The vector comprising a DNA sequence of interest operably linked to a regulatory region (enhancer/promoter sequences) can be directly introduced into appropriate host cells for expression of the DNA sequence of interest without further cloning.

For expression of the DNA sequence of interest in mammalian host cells, a variety of regulatory regions can be used, for example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter. Inducible promoters that may be useful in mammalian cells include but are not limited to those associated with the metallothi- onein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), β-interferon gene, and hsp70 gene. It may be advantageous to use heat shock promoters or stress promoters to drive expression of the DNA sequence of interest in recombinant host cells.

In addition, the expression vector may contain a selectable or screenable marker gene for initially isolating, identifying or tracking host cells that contain the vector.

A number of selection systems may be used for mammalian cells, including but not limited to the Herpes simplex virus thymidine kinase, hypoxanthine-guanine phos- phoribosyltransferase, and adenine phosphoribosyltransferase, genes can be em- ployed in tk ^' , hgprf or aprf cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neomycin phosphotransferase (neo), which confers resistance to the aminoglycoside G-418; and hygromycin phosphotransferase (hyg), which confers resistance to hygromycin. Other selectable markers, such as but not limited to histidinol and Zeocin® can also be used. EXPRESSION SYSTEMS AND HOST CELLS

For use with the methods of the invention, the host cell and/or the host organism preferably does not express the Bovine Papilloma Virus E1 protein. Furthermore, preferably the vector of the invention does not encode the Bovine Papilloma Virus E1 pro- tein.

Preferred mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, such as monkey kidney cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR; mouse Sertoli cells; mouse fibroblast cells (NIH-3T3), monkey kidney cells (CVI ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCC CCL51).

The vectors of the invention may be synthesized and assembled from known DNA sequences by well-known techniques in the art. The regulatory regions and enhancer elements can be of a variety of origins, both natural and synthetic. Some host cells may be obtained commercially.

The vectors of the invention containing a DNA sequence of interest can be introduced into the host cell by a variety of techniques known in the art, including but not limited to, for prokaryotic cells, bacterial transformation, and for eukaryotic cells, calcium phosphate mediated transfection, liposome-mediated transfection, electroporation, microinjection, regular injection into a tissue, hydrdynamic injection into the bloodstream, topical application (including in the composition of cremes or oinpments or using patches), and cell-penetrating peptide mediated delivery.

In a specific embodiment, cell lines that express the DNA sequence of the invention may be engineered by using a vector that contains a selectable marker. By way of example but not limitation, following the introduction of the vector, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the vector confers resistance to the selec- tion and optimally allows only cells that contain the vector with the selectable marker to grow in culture.

GENE THERAPY APPROACHES

In a specifically preferred embodiment of the present invention, a vector of the invention comprising an expression cassette comprising DNA sequences of interest is administered to treat, or prevent various diseases. The DNA sequence of interest may encode a macromolecule, such as, but not limited to, a protein and/or a biologically active RNA molecule. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible DNA sequence, i.e., the DNA sequence produces its encoded protein or RNA molecule that mediates a therapeutic effect.

A non-integrating expression vector according to the present invention is a non- integrating viral vector, i.e. the vector is maintained in the nucleus without integrating into the chromosomal DNA, so that the expression of the foreign DNA/gene is transient. The vector lacks the essential components for the viruses' propagation but is attached to the host's chromatin and is therefor effectively distributed to the next generation of propagating cells, thus guaranteeing a stable but transient expression in the host. The viral vectors of the present invention have further been engineered for safe- ty by making them replication incompetent. Still further, the vectors of the present invention do not activate an innate immune response in the patient/host. It is their ability to efficiently but transiently infect cells and in this process to transfer DNA to the host without invoking an immune response that makes the expression vectors of the present invention particularly attractive as vectors for use in repeated administration reg- imens for gene therapy.

The expression vectors) of the present invention is in particular intended for use in a repeated administration regimen for non-intergrating gene therapy. The expression vector(s) of the present invention are used in a repeated administration regimen for non-integrating gene-therapy with a viral non-integrating vector.

Non-integrating gene transfer has the advantage that the expression of the gene of interest can be directely controlled by administration dose and regimen and also be terminated. Furthermore, there is a lesser risk for oncogenecity. On the other hand, long term and/or life long treatment with non-integrating gene therapies is often hampered by an ever increasing immunological response to the vector elements of non-human and/or trans-species origin. The expression vector(s) of the present invention is/are particularly well suited for a repeated administration in a gene therapy regimen because they do not activate an immunological response in the patient treated with the gene therapy, which prevents the development of adaptive immune responses for long term efficacy. The expression vector(s) of the present invention do(es) not activate an innate immune response in a mammalian cell, as is demonstrated in the experimental section, wherein in particular experiments 6 and 7 clearly disclose that the expression vector(s) of the present invention do(es) not induce humoral responses in an animal model. It is therefore concluded that the expression vector(s) of the present invention will not activate a significant induction of antigen specific lgG1 response in the patient treated with the gene therapy.

The present invention further relates to a non-integrating expression vector according to the present invention, for use as a medicament, such as for use in gene therapy, such as for use in a repeated administration regimen for gene therapy, such as for use in a repeated administration regimen for non-integrating gene therapy.

Some embodiments of the present invention relate to a non-integrating expression vector according to the present invention, for use as a carrier vector for a gene, genes, or a DNA sequence or DNA sequences of interest, such as for one or more genes, or a DNA sequence or DNA sequences encoding a protein or peptide of an antibody, a therapeutic agent, a macromolecular drug, or any combination thereof.

Some embodiments of the present invention relate to a non-integrating expression vector according to the present invention, for a use selected from the group consisting of treating and/or preventing an inherited or acquired genetic defect, for use in antibody and/or monoclonal antibody therapy, for use in enzyme replacement therapy, for use in therapy with antibody or monoclonal antibody fragments, for use in delivery of an acute medicament, for use in cancer treatment, for production of a therapeutic macromolecular agent in vivo and for treatment of viral infections. Any of the methods for gene therapy available in the art can be used according to the present invention. .Exemplary methods are described below.

For general reviews of the method of gene therapy, see, Goldspiel of a/., Clinical Pharmacy 12 (1993) 488-505; Wu and Wu, Biotherapy 3 (1991 ) 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993) 573-596; Mulligan, Science 260 (1993) 926- 932; Morgan and Anderson, Ann. Rev. Biochem. 62 (1993) 191-217; May, TIBTECH 1 , l(5) (1993)155-215. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel ef a/, (eds.), Current Proto- cols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

The following animal regulatory regions, which exhibit tissue specificity and have been utilized in transgenic animals, can be used for expression of the DNA sequence of in- terest in a particular tissue type: elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region which is active in pancreatic beta cells, immunoglobulin gene control region which is active in lymphoid cells, mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells, albumin gene control region which is active in the liver, alpha-fetoprotein gene control region which is active in the liver; alpha 1 -antitrypsin gene control region which is active in the liver, beta-globin gene control region which is active in myeloid cells; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain; myosin light chain-2 gene control region which is active in skeletal muscle, gonadotropic releasing hormone gene control region which is active in the hypothalamus, control region of a keratin, involucrin or loricrin gene which are active in keratinocytes, control region of dectin-2 gene which is active in Langerhans cells, and control region of Human Slow Troponin I or muscle creatine kinase which are active in muscle cells.

MONOCLONAL ANTIBODIES

In certain embodiments of the invention a vector of the invention contains a DNA sequence of interest that encodes an antibody, such as an mAb (monoclonal antibody). The antibodies expressed can be of natural, synthetically altered and or recombinant origin, such as but not limited to selected from the group consisting of bispecific antibodies, trispecific antibodies and nanobodies. As an alternative to recombinant protein administration, the expression vectors according to the present invention may provide a novel mean for systemic delivery of monoclonal antibodies. This can beenused a wide variety of pathological conditions, including but not limited to cancer, infectious diseases, drug addiction, retinal neovas- cularisation and Alzheimer's disease. The use of the expression vectors according to the present invention are safe and limit the therapeutic effect due to substantially reduced immune responses against viral antigens and high transduction efficiency. mAbs can be produced with recombinant technologies, allowing the design of fully human antibodies of any specificity and for diverse purposes. Recombinant antibodies can be engineered with optimized properties, such as antigen-binding affinity, molecu- lar architecture and dimerization state, and fused with a vast array of effector moieties to enhance their tumor-targeting ability and potency. The use of the herein described vectors for gene therapy offers additional benefits by achieving temporary but still sustained and effective concentrations of therapeutic antibodies directly at points of target intervention. This compensates for any rapid blood clearance of antibody fragments and could make the antibody less immunogenic and better tolerated. Furthermore, antibody molecules can thus be provided with new functions in unexpected scenarios: e.g. expression of antibody domains in precise intracellular locations and grafting of new binding activities to engineered cells. In particular, this is applicable for antibody- based cancer therapy. four main types of CmAbs: murine, chimeric, humanized and human CmAbs. Murine CmAbs, derived exclusively from mouse, were the first to be applied in cancer chemo- therapeutics. Utilization, however, was rapidly revoked because of inability to effectively interact with components of the human immune system due to their foreign na- ture and subsequent limited recognition by the host immune system. Chimeric CmAbs typically comprise variable regions derived from a murine source and constant regions (65%) derived from a human source. Chimeric CmAbs can also be non-humanized (chimeric trifunctional CmAbs), rat-mouse hybrid monoclonal antibodies that have three different antigen-binding specificities: for tumour cells, T lymphocyte cells and one for accessory cells. The development of chimeric CmAbs that possess a fully hu- man Fc portion provided considerably less immunogenic and more efficient interaction with human effector cells and the complement system than murine CmAbs. Humanized CmAbs are predominantly (90%) engineered from a human source with the exception that the complementarity-determining regions of the Fab portion are of murine origin; they are even less immunogenic than chimeric CmAbs. Human CmAbs, which are 100% human, are engineered from transgenic mice, and compared to chimeric and humanized CmAbs, have higher affinity values toward human antigens and minimal or no hypersensitivity responses. Chemotherapeutic monoclonal antibodies may be conjugated to other forms of cancer therapy and this facilitates greater efficacy. More importantly, conjugation provides targeted attack at cancer cells and therefore reduced widespread systemic toxicities to normal cells. There are three types of conjugated CmAbs: radiolabeled CmAbs which are linked to radionuclide particles, chemolabelled CmAbs which are attached to anti-neoplastic drugs and immunotoxin CmAbs which are attached to plant and bacterial toxins.

INHIBITORY ANTISENSE AND RIBOZYME

In certain embodiments of the invention a vector of the invention contains a DNA se- quence of interest that encodes an antisense or ribozyme RNA molecule. Techniques for the production and use of such molecules are well known to those of skill in the art.

Antisense RNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.

A sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with at least the non-polyA portion of an RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or tri- plex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. In other embodiments of the invention, the anti- sense nucleic acids are at least 100, at least 250, at least 500, and at least 1000 nucleotides in length. Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense DNA sequence are compared with those obtained using a control DNA sequence. It is preferred that the control DNA sequence is of approximately the same length as the test oligonucleotide and that the DNA sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

While antisense DNA sequences complementary to the target gene coding region sequence could be used, those complementary to the transcribed, untranslated region are most preferred.

For expression of the biologically active RNA, e.g., an antisense RNA molecule, from the vector of the invention the DNA sequence encoding the biologically active RNA molecule is operatively linked to a strong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of suffi- cient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation of the target gene mRMA. For example, a vector of the invention can be introduced, e.g., such that it is taken up by a cell and directs the transcription of an antisense RIMA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Ex- pression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region, the promoter contained in the 3 long terminal repeat of Rous sarcoma virus, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionein gene, ere.

In certain embodiments of the invention, a vector of the invention contains a DNA sequence, which encodes a ribozyme. Ribozyme molecules designed to catalytically cleave target gene mRMA transcripts can also be used to prevent translation of a tar- get gene mRNA and, therefore, expression of a target gene product (see, e.g., WO90/11364).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleo- lytic cleavage event. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Patent No. 5,093,246, which is incorporated herein by reference in its en- tirety.

While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy target gene mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA has the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art. Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target gene mRNA, i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts. The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA). The Cech-type ribozymes have an eight base pair active site, which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The invention encompasses those Cech- type ribozymes, which target eight base-pair active site sequences that are present in the target gene.

Expression of a ribozyme can be under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

In instances wherein the antisense and/or ribozyme molecules described herein are utilized to inhibit mutant gene expression, it is possible that the technique may so efficiently reduce or inhibit the translation of mRNA produced by normal target gene alleles that the possibility may arise wherein the concentration of normal target gene product present may be lower than is necessary for a normal phenotype. In such cases, to ensure that substantially normal levels of target gene activity are main- tained, therefore, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity may, be introduced into cells via gene therapy methods that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized. Alternatively, in instances whereby the target gene encodes an extracellular protein, it may be preferable to co- administer normal target gene protein in order to maintain the requisite level of target gene activity.

Methods of administering the ribozyme and antisense RNA molecules are well known in the art. PHARMACEUTICAL FORMULATIONS AND MODES OF ADMINISTRATION

In a preferred aspect, a pharmaceutical of the invention comprises a substantially purified vector of the invention (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject to whom the pharmaceutical is administered in the methods of the invention is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably a human.

In certain embodiments, the vector of the invention is directly administered in vivo, where the DNA sequence of interest is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art. The vectors of the invention can be administered so that the nucleic acid sequences become intracellular. The vectors of the invention can be administered by direct injection of naked DNA; use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); coating with lipids or cell-surface receptors or transfecting agents; encapsulation in microparticles or microcapsules; administration in linkage to a peptide which is known to enter the nucleus; administration in linkage to a ligand subject to receptor-mediated endocytosis (which can be used to target cell types specifically expressing the receptors); etc. In a specific embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome.

In certain embodiments, the vector of the invention is coated with lipids or cell-surface receptors or transfecting agents, or linked to a homeobox- like peptide which is known to enter the nucleus.

In certain other embodiments, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet other embodiments, the vector of the invention can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor.

Methods for use with the invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Methods for use with the invention further include administration by any con- venient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). In a specific embodiment, it may be desirable to administer a vector of the invention by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. Care must be taken to use materials to which the vector does not absorb. Administration can be systemic or local. In certain embodiments, a vector of the invention is administered together with other biologically active agents such as chemotherapeutic agents.

In yet another embodiment, methods for use with the invention include delivery via a controlled release system. In one embodiment, a pump may be used. In another em- bodiment, polymeric materials can be used.

Other controlled release systems are discussed in the review by Langer, Science 249 (1990)1527-1533. Pharmaceutical compositions of the invention comprise a therapeutically effective amount of a vector of the invention, and a suitable pharmaceutical vehicle. In a specific embodiment, the term "suitable pharmaceutical vehicle" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more par- ticulariy in humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such suitable pharmaceutical vehicles can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is ad- ministered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include stand- ard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the nudeic acid or protein of the invention, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a specific embodiment, the pharmaceutical of the invention is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intrave- nous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical of the invention may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for exam- pie, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical of the invention is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical of the invention is administered by injection, an am- poule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellent, e.g., dichlorodi- fluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be de- termined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The amount of a vector of the invention, which will be effective in the treatment or prevention of the indicated disease, can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the stage of indicated disease, and should be decided ac- cording to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The present invention may be better understood by reference to the following non- limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

EXAMPLE 1

The specific promoters were selected by literature search. Then the sequences were amplified from the source genomic DNA with specific primers. The primers were designed so that suitable specific restriction sites were added to the ends of the promoters to allow directed cloning into the GTU® in front of the GOI or E2.

Ubiquitous and tissue type specific GTU* vectors

USING SPECIFIC PROMOTERS FOR EXPRESSION OF E2 AND/OR GENE OF INTEREST

Ubiquitous promoters:

• CMV

• RSV LTR

• beta-actin

■ ubiquitin

As can be seen in figure 2, 10pg of the firefly luciferase and EGFP (linked by 2A peptide and encoded by a single cDNA), expressing epi-GTUK14 or "non-GTU" control was injected (five injection sites for both DNA) into the swine superficial skin by insulin syringe. In the epi-GTUK14, a human keratin 14 (hK14) promoter was used as an ex- pression controlling element in front of both E2 and EGFP-2A-luc2 markergene. The "non-GTU" vector was identical to the epi-GTUK14, but contained disrupting mutations in the E2 coding sequence, thus the E2 protein is not produced from the mutated gene of the "non-GTU". 48h later, the transfected region was visually identified by EGFP expression using blue flashlight with appropriate filters. Thereafter, the EGFP positive region in each site was collected using 8mm punch biopsies. The biopsies were lysed in IxCCLR buffer (Promega) and luciferase activities were measured from the lysates using Luciferase Assay System kit (Promega). As is shown in figure 3, the experiments are executed as described for figure 2, but here 20 pg of epi-GTUK14 was used with either equal molarity or weight of the regular K14 vector. As the regular vector has a size ~1.6 times smaller than epi-GTUK14, there is 1.6 times more plasmid molecules for the same mass of regular K14 vector than for the epi-GTUK14. Despite of this, the results demonstrated the beneficial expression properties of the epi-GTUK14 over regular K14 in both case.

The experiment shown in figure 4 was performed as described for figure 2, but here 20 pg of epi-GTUK14 or "non-GTU" was used and the luc expression properties were analyzed in timeline. Samples were collected at different time points (24, 48 and 72h) after delivery, as indicated. The data was expressed as ratio of mean values epiG- TUK14/"non-GTU" ("fold activation"). The results showed that the benefit of the epi- GTU® increases in time. Figure 5 shows the results of immunofluorescence staining of the frozen sections prepared from the swine skin biopsy. The skin was injected as described for figure 2. However, ubiquitous GTU® expressing artificial MultiHIV antigen was used (CMV- MultiHIV, RSV LTR - E2). Antibodies conjugated with different fluorochromes were used (anti - HIV gag (included into the MultiHIV) conjugated with Alexa568, anti-E2 conjugated with Alexa488).

EXAMPLE 2

Figure 6 shows the outcome of an Ichor experiment. The GTU® and control regular plasmid (plMS-431) were used, expressing a secretable alkaline phosphatase (mSEAP). The enzymatic activity of the SEAP in the blood (vertical axis) was meas- ured in time after DNA delivery, as shown in the horizontal axis. IM is most likely intramuscular injection and in the TDS-IM groups, the injection is assisted by electro- poration, using an Ichor device.

As can be seen in figure 7, GTU® and non-GTU® control vectors were designed and constructed. All vectors contained a tricistronic GEF markergene, expressing three marker proteins from a single cDNA:

1. secretable Gaussia luciferase (Glue) measurable in blood samples;

2. EGFP detectable by fluorescence using in vivo imaging; 3. intracellular firefly luciferase that can be measured in muscle cell extracts or by in vivo imaging with luciferine.

Both the markergene and E2 expression were controlled by muscle specific promoter 3xUSE/Tnl (human Slow Troponin I promoter (Tnl) and its oligomerized upstream enhancer elements (USE), described in Blain et al. 2010 [PMID: 19719387]). The "non- GTU" was a regular vector containing the same expression unit for GEF but no E2 cassette or E2 BSs. Equimolar amounts (40 pg GTU® or 29 ug non-GTU) were delivered to muscle tibialis anterior of Swiss Webster mice (22-25 g), using 5 mice in both group:

25 μΙ hyaluronidase (400 U/ml) solution was injected into the delivery site;

30 μΙ DMA was injected into the single muscle; electroporation was administered just after DNA delivery (NEPA21 with tweezer-electrodes and conditions: 2 x 4 pulses in different orientations, 75 V, pulse length = 20 ms, gap between pulses = 80 ms).

The expression from the delivered plasmids was quantitatively analyzed by Glue activity in 5 μΙ whole peripheral blood from tale tip bleeding that were collected in time points 3, 7 and 10 days after DNA delivery. The collected blood was immediately mixed with 1 pi of 50 mM EDTA pH 8.0 and stored at -70 °C until measurement of Glue activity. The Glue assay was conducted as next. The blood of all time-points were analyzed together the samples were melted by mixing with 50 μΙ of BioLux GLuc Assay Buffer (Biolabs) and then incubated 10-12 min at room temperature. Thereafter 0.5 μΙ of 100x BioLux* GLuc Substrate (Biolabs) was added to the sample and mixed by vortexing 1-2 sec. Then the signal was immediately measured as relative light units (rlu) using Glomax 20/20 luminometer (Promega) with integration time = 10 sec.

There are group mean values with SD in time in the upper diagram of fig. 7 and data from individual animals in 3 and 7 days' time points.

In this example it should be noted that both GTU® and non-GTU® signals were silenced shortly after DNA delivery due to immunological mechanisms against the GEF product and long-term monitoring was not possible. Results of expression in MUSCLE: GTU ^® vs "non-GTU"

^■ GTU® expression enhancement function is detectable in mouse muscle in

vivo;

• Widely used DNA delivery by electroporation is compatible with GTU® usage.

Conclusion:

As can be seen in figures 6 and 7, using specific promoters for expression of gene of interest and E2, families of ubiquitous and tissue specific gtus have been developed. GTU* has superior expression properties over regular vectors in vivo in different target tissues (epidermis, muscle);

EXAMPLE 3

Based on results of the previous study (referred in figure 7), the experiments shown in figure 8 were designed for further characterization of the key factors for GTU® func- tionality (expression enhancement) in skeletal muscle cells.

Instead of employing a surrogate marker, actual potential therapeutic molecules (antibodies) are expressed as gene of interest from the GTU® or the non-GTU® control. Six different vectors were constructed expressing intact mouse lgG2a antibody 5c2 against human (and also mouse) MANF protein. This way the induction of anti-product immune response was avoided that usually allows only short-term production/activity of the foreign protein (luciferase) tested in previous in vivo studies. Both heavy (HC) and light chain (LC) of the antibody is expressed from the single cDNA transcribed from e 3xUSE/Tnl promoter. The heavy and light chain was separated by FMDV 2A peptide sequence + furin site resulting in secretion and assembly of antibody com- prised of natural heavy chain (HC) and light chain (LC) products.

The animals and delivery were as described for figure 7 above. There were 6 mice per group. The expression from the delivered plasmids was quantitatively analyzed by MANF ELISA in peripheral blood. For sampling, 1 :20 diluted blood samples were collected at different time points by mixing 5 μΙ of blood from tale tip cuts with 95 μΙ of PBS. The samples were stored at -20°C until analysis. The analysis time points were collected in the next time-points: 0 (baseline before DNA delivery), 3 days, 1 , 2, 3, 4, 5, 6, 8 and 12 weeks. All time points were analyzed by pooling the samples (dilution 1 :160) in the group.

Figure 9 shows the same experiment. Scatter blots of the individual samples in time- points 4, 6, 8, and 12 weeks. Statistically significant differences between GTLKB ⁾ and regular groups are shown by asterisks.

Figure 10 lower diagram shows (muscle GTU): in time data of individual animals of the same set as above (3xUSE/Tnl, controlling both the E2 and antibody expression). Anti-MANF MAb concentration in the blood was quantified in the peak level. In the upper diagram it is shown: Same but "ubiquitous GTU" was delivered. In this GTU®, the E2 expression was controlled by the 3xUSE/Tnl, but antibody expression was under control of a strong ubiquitous CAG promoter. Immunogenic marker vs natural protein products

Using the foreign protein (e.g. Luciferase) as marker expressed from the GTU®, the expression is rapidly silenced by immunological mechanisms; Thus, using natural intrinsic (non-immunogenic) proteins as markers is needed for analysing the long term expression.

Conclusion:

As can be seen in figures 8-10, Intramuscular delivery of GTU® expressing monoclonal antibody (as non-immunogenic intrinsic protein) results in therapeutically relevant concentrations of the antibody in the bloodstream over prolonged period;

EXAMPLE 4

gtGTU* vectors

gtGTU® vectors are designed based on minimizing the plasmid vector size and removal of dispensable components (e.g. bacterial origin, antibiotic resistance marker) which in itself improves the vector outcome in gene therapy applications in vivo. Higher and more prolonged expression of the transgene could be achieved. Further, the bulk of immunostimulatory sequences (ISSs) were also removed, striving to completely remove all ISSs in gene therapy vectors. This reduces the ability of activation of in- nate immune responses and thus decreases the risk of the vector itself to work as a DNA vaccine which potentially can induce immunity against the product of interest, especially when administered repetitively. Requirements for the vector in DNA vaccination versus gene therapy applications are different. The gtGTU® concept was specifically created for the usage of GTU* in gene therapy applications. It includes minimizing the vector elements, e.g. incorporating the E2BSs into the intron, removal of dispensable components, e.g. bacterial origin, antibiotic resistance marker ensures more efficient delivery/expression. The gtGTU® concept also includes removing a bulk of immunostimulatory sequences (ISSs) from the GTU* DNA sequences by intelligent mutagenesis. ISS removal reduces the ability of activation of innate immune responses and thus decreases the risk of the vector itself to work as a DNA vaccine, which can induce immunity against the product of interest, especially when administered repetitively. For example, ISS-free variants of several promoters are designed (based on RSV LTR, CMV, for example), as well as polyadenylation signals, introns etc. for their usage in gtGTU® vectors.

The general gtGTU® concept is disclosed in figure 11. The gtGTU® concept includes minimizing the plasmid vector size and removing potentially immunostimulatory se- quences. As is demonstrated, for the design and construction of gtGTU® vectors, whole sequences of GTU* gene vaccination vector (GTU8) were completely redesigned to avoid mouse or human ISSs. In addition, the final gtGTU® products were produced in most compact form as minicircle plasmids. In detail, three different gtGTU® configurations were designed and constructed that express the Glue markergene. The first configuration (gtGTU 1 on figure 12) generally mimics the structure of the latest generation of GTU® gene vaccination vector (GTU8). However, the gtGTU 1 differs from GTU8 by absence of bacterial backbone sequences (resistance marker, on) and by absence of ISSs (GTU8-Gluc has 16 human ISSs and 36 mouse ISSs). In gtGTU 1 a 3' intron sequence is used for expression of gene of interest and E2 binding sites (E2BSs) are located in front of its promoter (CMV).

In contrast, the 3' rabbit intron is replaced with synthetic 5' intron and the E2BSs were placed inside of the designed ISS-free synthetic intron in second and third configura- tions (gtGTU2 and gtGTU3 in fkjurel) for the purpose to make the vector even more compact. The difference between gtGTU2 and gtGTU3 is only in the orientation of the E2 gene (figure 1) which has been demonstrated to be an important factor for durability of expression from the vector in some systems. Thus, all these initial gtGTUs were designed so that these: all human ISSs (TCGTW) and mouse ISSs (RRCGYY) are removed by point mutations; these express the Glue markergene as gene of interest. Figure 12 shows schemes of three gtGTU® configurations designed. Coding sequences for Glue and E2 are indicated by red colour. As mentioned above, all mouse and human ISSs were eliminated in gtGTUs by point mutations. The mutations were also made in promoter and polyadenylation regions as well as in introns. The sequences in these regions contain structural elements and binding sites for cellular factors that are important for transcription of the gene of interest and E2. Generally, in the process of ISSs removal, all crucial elements (TATA, CAAT, Inr, SD, SA, pA signals etc.) were kept intact. However, the CMV promoter contains five 19-bp elements that may be important for transcription. Four of them are identical and recognized as cAMP response elements (CRE) but contain mouse ISS in their core. The fifth 19-bp element is a variant of CRE (AF-1/CRE) and it does not contain ISS (Keller et al., J Virol 77, 6666). Thus we used strategy by which all four ISS-containing 19-bp elements were replaced with AF-1/CRE elements in the gtGTU® vector. Some point mutations were also made in E2 gene that is controlled by RSV LTR promoter. In order to evaluate the activity of gtGTU® configuration, pMA- gtGTU1-Gluc was initially tested in transient expression assay. The evaluation was made in comparison with original gene vaccination vector GTU8 expressing the Glue markergene (GTU8-moptGluc). The regular pCMV vector (pCMV-moptGluc) contains the same Glue gene as GTU8-moptGluc but not the E2 gene was also included.

In particular, the pMA-gtGTU1-Gluc was transfected into the cultured human RD (rhabdomyosarcoma derived) cells using 3 different DNA amounts. Equimolar amounts of GTU8-moptGluc and pCMV-moptGluc were used as controls. Twenty hours and 44h post-transfection Glue activity was measured from the culture medium (figure 13). The cells were lysed at 44h time-point and expression of E2 in cells was analysed by Western blot (figure 14). As it is shown in figure 13, the Glue activities in supematants of cells transfected with pMA-gtGTU-Gluc were comparable with these observed in cultures transfected with the GTU8-moptGluc. Both GTU8-moptGluc and pMA-gtGTU1-Gluc plasmids demonstrated higher expression levels than pCMV-moptGluc which indicates the E2 and E2BSs mediated expression enhancement as function of the GTU* system. Direct analysis of E2 expression by Western blot (figure 14) showed E2 expression both from GTU8 and pMA-gtGTU1 vectors. No expression was observed from pCMV- moptGluc (negative control, does not contain E2 gene). Expression of E2 was somewhat higher from pMA-gtGTU1 than from GTU8-moptGluc. That is probably caused by replacement of some infrequently used codons in E2 cDNA with more frequently used variants during the design of gtGTUI sequence. In conclusion, the expression activities of expression units for E2 and gene of interest as well as GTU® function (transcription enhancement) was not hampered during ISS removal in design of gtGTU® sequence.

EXAMPLE 5

Preclinical efficacy of in vivo treatment with gtGTU® vectors in anti-cancer model

Example 5 shows in vivo results of gtGTU® vectors being applied as vehicle for a therapeutic protein known from approved clinical approaches. An anti-cancer monoclonal antibody (MAb) was selected for these studies, in particular, bevaci- zumab/A.4.6.1. Bevacizumab (trade name: Avastin) is a recombinant humanized monoclonal antibody that is an angiogenesis inhibitor which slows the growth of new blood vessels (neovascularization) by inhibiting human vascular endothelial growth factor A (VEGF-A). It is clinically approved for treatment of advanced cancers which growth is depending on neovascularization process. The usable mouse models include intraperitoneal tumor induced by human ovarian cancer cell line OV-90 and Ewing sarcoma A-673 model, for example. In Example 5, human tumors were established as xenografts in immunocompromised mice to demonstrate the improved therapetic ability of anti-cancer antibody expression from gtGTU® vectors. Study design

Immunocompromised mice (thus having susceptibility to human tumor xenografts) were transfected with gtGTU(i) or non-GTU® control plasmids expressing heavy and light chain of mouse MAb A4.6.1. that can block its antigen Human Vascular Endothe- lial Growth Factor A (hVEGF-A), and therefore its function in promotion of neovascularization. After establishment of peak level of MAb in blood, the mice were inoculated with hVEGF-A expressing Ewing sarcoma A673 cells. To assess if gtGTU® expression of A.4.6.1. MAb specifically suppresses the growth of the tumors, the tumor growth was monitored in mice with different treatments, including positive and nega- tive controls: e.g. administration the A.4.6.1. as protein or administration of gtGTU® expressing non-relevant MAb 8E2 (against E7 protein of a human papillomavirus).

Materials and Methods

gtGTUs and pReg

Figure 24 and SEQ.ID.NOs: 19-21 show the schematic maps and the DNA sequences of the vectors that were used in Example 5, which are described below. The gtGTU® vectors use comprise DNasel hypersensitive srte1 (HS1) beta globin chromatin insulator elements in the ends of the bacterial backbone. These are intended to protect transgene cassettes from silencing and positional effects in muscle cells. gtGTUM(i)-A461(SEQ.ID.NO.:19)

gtGTUM(i)-A461 expresses the A.4.6.1. MAb cDNA consisting of mouse codon optimized sequence encoding the lgG1 heavy chain (HC) and kappa light chain (LC) pre- proteins separated by 2A peptide of foot and mouth disease virus (HC-2A-LC). There are also furin cleavage sites engineered between the C-terminus of HC and N- terminus of the 2A peptide. gtGTUM(i)-A461 HC (SEQ.ID.NO.:20) and GTUM(i)-A461 LC (SEQ.ID.NO.:21) The A.4.6.1. MAb is expressed from two gtGTU(i) plasmids mixed (mass ratio 1 :1). gtGTUM(i)-A461HC expresses lgG1 HC of A.4.6.1. and GTUM(i)-A461LC expresses kappa LC of A.4.6.1. gtGTUM(i)-8E2 This vector is analogous to gtGTUM(i)-A461 , but expresses supposed non-relevant mouse MAb 8E2 directed against HPV18 E7 protein. pReg(i)-A46

This vector is a regular, non-GTU® plasmid expressing the A.4.6.1 and containing similar elements as gtGTUM(i)-A461 , but not the E2 expression cassette.

Proteins

A.4.6.1. MAb and 8E2 MAb proteins were produced in CHO (CH085EBNALT, lcosagen) cells and purified using protein-G binding and gel filtration steps. Finally, the buffer was changed to PBS pH 7.4 (cone.1 ,25 mg/ml), the proteins were aliquoted and stored in -20 C. The MAb binding activity of purified proteins were confirmed in VEGF and E7 ELISA, respectively. hVEGF-165 and HPV18E7 proteins used as antigens in ELISA assays were produced in CHO (CH085EBNALT, lcosagen) cells and purified via His-tag. Animals and study groups

The size of and composition of each of the groups is represented in Table 1.

DNA delivery DNA

For delivery of gtGTU® and pReg vectors, NEPA21 device with CUY650-P5 electrodes was used. The conditions were pre-optimized in earlier studies. One hour before DNA injection, 30 pi hyaluronidase (0.5 mg/ml, prepared from Type IV-S, powder (750-3000 units/mg, Sigma-Aldrich H4272) solution in sterile endotoxin-free PBS was injected into the delivery site. The DNA (cone = 1.5 mg/ml) delivery was by plain injec- tion into tibialis anterior of both rear limbs using 30 pg endotoxin free DNA solution in 30 μΙ of PBS (30 μΙ/limb = 45pg/limb = 90 pg/animal). All injections were done using insulin syringes with 29g (0.3 mm) attached needle. EP pulses were administered just after DNA delivery as shown in Table 2.

Protein

Groups G5 and G6 were treated with A.4.6.1. or control MAb 8E2, respectively. Ac- cording to the Presta et al. 1997, MAbs were administered intraperitoneally (i.p.) at the dose of 5 mg/kg, twice weekly, starting 24 h after tumor cell inoculation, (every Thursday and Monday, presented in study timeline in Fig. 2. MAb cone. = 1 ,25 mg/ml→ 120 μΙ (~30 g body weight).

Tumor induction and monitoring

A673 Ewing sarcoma tumors were induced 30 days after DNA delivery according to the timeline represented in Fig 25. This was performed essentially as described in Gerber et al., 2000: Human A673 rhabdomyosarcoma cells were trypsinized and trypsin was inactivated with fetal bovine serum containing medium. Then the cells were precipitated and re-suspended in ice-cold PBS. After second precipitation, the cells were re-suspended in DMEM medium with high glycose, glutamax, pyruvate (Thermo Fisher Scientific 11995073). Next, the equal volume of Corning® MatrigeKD Matrix (solubilized basement membrane preparation, Coming 354262) was added to the cells. Then 100 μΙ of the mixture containing 4.6x10 ⁶ was subcutaneously injected per mice into the dorsal flank region.

Tumor size measurements were initially performed weekly, but more frequently in the end of the study (Fig.25) measuring through skin. Tumor volume was calculated using the ellipsoid volume formula ττ/6 x L x W x H, where L = length, W = width, and H = height (Tomayko and Reynolds, 1989). Animals were killed using cutoff when tumors reached≥2500 mm ³ (as used previously for the model, see Eshun 2010) or at day 30 after tumor induction (Fig. 25), whichever came first. Thus, the animals with tumors ≥2500 mm3 were considered as non-survivals in the analyses. Blood sampling and antibody concentration measurements

For sampling, 1 : 20 diluted blood samples were collected from each animal at different time points as described in Fig. 25. Five microliters of blood from tale tip cut was mixed with 95 μΙ of PBS and the samples are stored at -20 °C until to analysis.

A.4.6.1. and 8E2 concentrations in blood were analyzed using 20 or 200 times diluted samples and purified protein standards for curve fitting in ELISA assays worked out in earlier studies.

Objectives

The Main objectives of Example 5 were as follows:

1. To elucidate whether A4.6.1. MAb delivery via gene based therapy using gtG- TU® vector can inhibit A673 tumor growth.

2. To analyze the A4.6.1. MAb production levels and/or kinetics from gtGTU® vector vs. non-GTU® control vector (administered in equal weight). If there is significant GTU>non-GTU® difference, it can be considered as confirmation of gtGTU® vector superiority for in vivo applications.

3. To analyze the A4.6.1. MAb production levels and/or kinetics using single gtG- TU® expressing HC and LC of the MAb or mixture of 2 gtGTUs expressing either HC or LC.

Results

Model validation

To validate the model in a particular study, tumor growth was analyzed in G5 and G6 groups subjected to intraperitoneal (i.p.) systemic protein administration of the antitumor MAb A.4.6.1. (G5) or negative isotype control MAb 8E2 (G6). First it was con- firmed that i.p administration of the MAb rapidly resulted in high MAb levels (tens of micrograms per ml) in blood in all animals treated (data not shown).

When tumor growth was analyzed, it was observed that the tumors were specifically inhibited by i.p. administration of the A.4.6.1. protein but not the 8E2 protein (Fig. 27). Similarly, clear distinctive tumor inhibition rates were observed in G1-G4 animals which received the gtGTU® or pReg DNA expressing the A.4.6.1. or 8E2 MAb (Fig. 28).

Confirmation of MAb production after DNA delivery

A4.6.1. and 8E2 MAb expression after DNA delivery was confirmed by blood samples collected 2 weeks after DNA delivery. For all animals, there was significant increase in ELISA readouts between baseline blood and 2 week timepoint, indicating secretion of the MAb by muscle cells (Fig. 29, left). Calculating the blood concentrations of the A.4.6.1. MAb in 2 weeks time point resulted values in range of 0.7 to > 10 pg/ml. The levels were higher in G2 where the A.4.6.1 HC and LC were expressed from the different GTU® plasmids. However, this group showed also the highest variability. The 8E2 MAb concentrations in group G3 reached from 0.7 to 1.3 pg/ml.

Thus, it was concluded that MAb expression was induced in all animals treated with DNA. Also it was concluded that expressing the HC and LC of the MAb from 2 gtGTU® vectors result in higher antibody concentrations in blood in early time-points when compared to expression from single gtGTU®.

Inhibition of tumor growth by delivery of gtGTU® vectors expressing A4.6.1. MAb

Survival curves were analyzed after tumor induction. Here, the mice with subcutaneous tumors reaching the size≥ 2500 mm3 were considered as non-survivals. Similar methodology is recognized also by other investigators (e.g., Eshun et al. 2010). Comparing the G1-G4 survival curves (Figure 30, upper) showed significantly better survival of G1 and G2 animals receiving gtGTU® DNA expressing the A.4.6.1. Interestingly, almost identical lower survival was observed for negative control group expressing non-relevant MAb 8E2 and G4 treated with A.4.6.1. expressing non-GTU® vector. As seen in lower diagram in Fig. 30, 23 days after tumor induction all animals were survived in GTU® groups G1 and G2, but most animals in G3 and G4 were already reached the cut-off (Fig. 30 lower).

Looking at the tumor growth curves of G1-G4 females in Fig. 28, sorting the animals by received treatment showed the distribution of each animal into one of the 3 tumor growth characteristics: 1) non-inhibited growth; 2) growth inhibited 21 days; 3) growth inhibited 30 days (until end of the study). Although there was no exact correspondence between treatment and tumor growth characteristic, some significant correlations were possible to find. In sharp contrast to G3 (neg. control MAb expressing gtGTU) and G4 (non-GTU® expressing A.4.6.1.), there was tumor growth inhibition observed more than 50% of animals in groups G1 and G2 expressing the A.4.6.1. MAb from gtGTU® in 21 and 23 days after injection of A673 cells (Fig. 31).

Tumor growth rates (% of growth) were analyzed in 2. week (between days 7 and 14) and 3. week (between days 14 and 21) after injection of the A673 cells (all animals were alive and measured at day 21) based on calculated tumor volumes. As seen in Fig. 32 (left), the growth was slower for groups G1 and G2 expressing A.4.6.1. MAb. Only for these groups the shrink of the tumors (negative growth) was observed. Statistical analysis (unpaired t-test, two-tailed) showed statistical significance between growth rates of gtGTU® A.4.6.1. groups (G1 +G2) compared to G3 (neg. MAb gtGTU) or G4 (non-GTU® A4.6.1). Similarly, there was statistically significant difference in tumor growth rate in week 3, when G1+G2 vs. G4 was compared (Fig. 32, right).

Conclusion

It was concluded that there were improved survival and reduced tumor growth rates for gtGTU® groups expressing A.4.6.1 MAb compared to animals expressing the negative control MAb from gtGTU® or A.4.6.1. MAb from the non-GTU® vector.

A4.6.1. MAb expression kinetics

Measuring the A.4.6.1. blood concentrations also in later timepoints (5, 6 and 8 weeks) after DNA delivery indicated that sharp difference in MAb production for single GTU® (HC-LC) vs. 2 GTUs (HC and LC) that was observed in 2 week time point (Fig. 33) was not the same in later time points (Fig. 34). Instead the expression levels showed rather minor differences between the groups. In all groups the maximal A.4.6.1. blood levels were observed in week 5-6. In addition, there was statistically significant gtGTU® > non-GTU® difference (unpaired t-test, two-tailed) in MAb expression level in 8 week time point (data not shown).

Conclusions from Example 5

Expressing the MAb from 2 gtGTU® vectors instead of single gtGTU®, results in higher antibody concentrations in blood in early time-points but no significant difference later (5 week and after this).

In later time-points there was statistically significant higher anti-cancer MAb expression from gtGTU® vector compared to non-GTU® control administered in equal weight (molar excess).

Thus, significant improvement in survival and decrease in tumor growth rates was found for anti-cancer MAb treatment using gtGTU® compared to gtGTU® treatment with negative control MAb.

EXAMPLE 6

Analysis of immunostimulatory properties of GTU® and gtGTU® vectors Objective

To analyze differences in innate immunity stimulation potential of GTU® vs gtGTU® vectors.

Rationale

Reducing ISSs reduces the ability of activation of innate immune responses and thus decreases the risk of the vector itself to be effective as a DNA vaccine, which can potentially induce immunity against the product of interest, especially when administered repetitively. The gtGTU* vector system was designed based on the GTU* platform which vectors were intended for and prior used as DNA vaccine, by mutational re- moval and/or modification of ISS.

In this example, the innate immune response stimulation was directly measured via changes of serum levels of marker cytokines IL-6 and TNF- a after GTU® or gtGTU® delivery via intraperitoneal (i.p.) administration. It has prior been demonstrated that in- jecting mice i.p. (intraperitonally) with 500 CpG-free plasmid DNA (e.g. pCpGfree- mcs from Invivogen) induced negligible amounts of TNF-a, whereas CpG-rich plasmid DNA induced significant amounts of TNF-a. All DNAs injected were endotoxin-free. A similar approach is herein used to test if ISS reduced gtGTU® induces less innate immune response than GTU. Different DMA amounts were used as well as another marker of CpG for induced innate immunity, IL-6, was included. DNAs tested

Materials and Methods

Plasmid DNAs were prepared as endotoxin free DNA (Favorgen Midi, EF) solved in Gibco EF DPBS, cat no. 14190, cone. = 900 Mg/ml. 5 and 50-times dilutions were mad in Gibco EF DPBS resulting concentrations 180 and 18 pg/ml, respectively

E. coli genomic DNA was purified from DH5a strain:

1. Bacteria from 100 ml of overnight culture (2YT medium) was lysed in 80 ml (20 mM TrisHCI pH8; 10 mM EDTA pH8; 100 mM NaCI; 0,2% SDS, Proteinase K 300 pg/ml, RNaseA 20 pg/ml), 55 °C, ON

2. precipitation with 2.5 vol ethanol in RT, collection of DNA with glass rod and 70% ethanol wash (immersed into)

3. 2 times phenol/chloroform + 1 chloroform extraction

4. Precipitation with NhUAc (1/5 of saturated solution) and 2.5 vol ethanol

5. 2 times wash with 70% ethanol

6. Diluted in Gibco EF DPBS, cat no. 14190, c = 900 pg/ml

Mice and administration

32 Balb/c, females, 7-12 weeks old

560 μΙ of DNA in PBS was injected i.p.

Sample collection

Two (2) time points:

1. base (before injection)

2. Four (4) hours after injection

In both time points, S-times diluted total blood samples were collected:

20 μΙ tail vein blood + 80 μΙ Reagent Diluent (DuoSet ELISA Ancillary Reagent Kit 2, R&D Systems DY008), stored at -20 C Additional serum samples in terminal 4h time point:

Serum samples are collected (as much as possible) from all animals, stored at -20 C

Analyses of total blood samples

Assays were conducted using:

1. Mouse TNF-alpha DuoSet ELISA kit (R&D Systems DY410-05) with DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems DY008)

2. Mouse IL-6 DuoSet ELISA kit (R&D Systems DY406-05) with DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems DY008)

According to manufacturer instructions using 50 μΙ of 5-times diluted total blood sam- pie per well

Results

The results are summarized in figures 34-37.

1. Figure 36 summarizes that all plasmid DNA delivered animals showed no significant elevation of the IL6 levels

2. Very high IL6 response (over highest standard point, thus > 5 ng/ml) after E. coli gDNA delivery (G8)

1. Figure 37 summerizes that all plasmid DNA delivered animals showed no significant elevation of the TNFa levels

2. TNFa induction from ~130 pg/ml in baseline to > 400 pg/ml was observed after E. coli gDNA delivery

Analyses of 4 h time-point serum samples 4h serums were diluted 2 (G1-G8 for TNFa and G1-7 for IL6 assay) or 50 (G8 for IL6 assay) times in Reagent Diluent (DuoSet ELISA Ancillary Reagent Kit 2, R&D Systems DY008) for TNF-a and IL-6 assays. Assays were conducted using:

1. Mouse TNF-alpha DuoSet ELISA kit (R&D Systems DY410-05) with DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems DY008)

2. Mouse IL-6 DuoSet ELISA kit (R&D Systems DY406-05) with DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems DY008)

According to manufacturer instructions using 50 μΙ of serum sample per well.

Results

The results are summarized in figures 38-41.

1. Figure 40 summerizes that all plasmid DMA received animals showed similar and low IL6 serum levels in 4h time-point.

2. Very high IL6 serum levels (equal or over highest standard point, thus >= 50 ng/ml in serum) were observed after E. coli gDNA delivery (G8).

1. Figure 41 summerizes that all plasmid DNA received animals showed similar and low TNFa serum levels in 4h time-point

2. Higher TNFa serum levels 400-1000 pg/ml were observed after E. coli gDNA delivery.

Conclusions

1. There was no significant induction of innate immune response markers IL-6 and TNFa after delivery of CpG-free, GTLKS ⁾ or gtGTU® plasmid

2. In contrast, E. coli (actually not endotoxin-free) gDNA induces high responses.

EXAMPLE 7

Analysis of immunostimulatory properties of GTU® and gtGTU® vectors humoral Im- mune response analysis Objective

To show differences in innate immunity stimulation potential of GTU® vs gtGTU® vectors.

Background and rationale

Reducing ISSs reduces the ability of activation of innate immune responses and thus decreases the risk of the vector itself to be effective as a DNA vaccine, which can potentially induce immunity against the product of interest, especially when administered repetitively. The gtGTU* vector system was designed based on the GTU* platform which vectors were intended for and prior used as DNA vaccine, by mutational re- moval and/or modification of ISS.

Several attempts were prior made to experimentally confirm that ISS reduced gtGTU® induces less innate immune response using direct measurement of marker cytokines IFN-a, IL-6 and TNF- a induction after GTU® or gtGTU® delivery. However, there was no significant induction of innate immune response markers IL-6 and TNFa after delivery of CpG-free, GTU® or gtGTU® plasmid. ([Experiments not shown/see example 6)

It is well known that CpG immunostimulatory sequences promote Th1 type immune responses over Th2 response with a number of different antigens. The Th1 biased immune response can be analyzed by ratio of antigen specific lgG1 (Th2) to lgG2a (Th1). Thus, mice were immunized with ovalbumin (OVA) as antigen alone or mixed with GTU®, gtGTU® or CpG oligodeoxynucleotide (ODN) as adjuvant; and OVA specific lgG1 and lgG2a response was analyzed at different time points.

Materials and Methods

Ovalbumin antigen:

EndoGrade® Ovalbumin (Item no. 321001, Hyglos GmbH) DNA adjuvants:

gtGTUM(i)-null#27 and GTUM(i)-null#89 (Figure 42)

Prepared as Endotoxin free DNA (NucleoBond® Xtra Maxi EF, Macherey Nagel) in Gibco EF DPBS (Thermo Fischer Scientific, cat no. 14190) mg/ml CpG ODN:

Class C CpG oligodeoxynucleotides ODN 2395 (Invivogen, tlrl-2395)

Mice:

Balb/cAnNCrl female mice, origin Charles River Laboratory, further breeding's are made in University of Tartu, Laboratory Animal Centre (generation 6), 6-7 weeks old:

Group G1

5

Immunizations

Mixtures injected intraperitoneally

Timeline

The study procedures in time are summarized in figure 43.

5 Blood samples collection

In each bleed time points (indicated in Timeline above, 0 days means base sample collected before 1. immunization), 20-times diluted total blood samples were collected: 5 μΙ of peripheral blood (from tail tip bleeding) was mixed with 95 μΙ of 1%BSA-PBS; and the samples were stored at -20 °C up to analyses.

10

OVA ELISA

RESULTS

Analysis of pooled samples

First, dilution curves were analysed for pooled samples of each group in each time point using OVA ELISA with lgG1 or lgG2a specific secondary antibody. The objectives were to elucidate: i) if and from which time-point the OVA-specific Abs are detectable; and ii) to find optimal dilution for analysis of individual samples.

As illustrated in figures 44-47, OVA specific lgG1 and lgG2a antibodies are detectable not earlier than 17 and 22 days after initial immunization (or after 4. and 5. immunization, respectively). Not surprisingly, lgG2a (Th1) response was induced mainly in positive control group G3 immunized with CpG ODN adjuvant (figures 45 and 47). However, completely unexpected was to detect the strongest lgG1 response shown in G4 (gtGTU) group (figure 46).

Also, based on pool analyses, blood dilution 1 :1000 was considered as most optimal for analysis of individual samples

Analysis of individual samples

Based on pooled samples analysis, only latest time-point (22 days) samples were analysed in level of individual animals (blood diluted 1 :1000) using same ELISA assay as described for pools. In figure 47, it is clearly seen that: i) lgG1 response in gtGTU® group is created by one highly outlier animal; and ii) actually only G3 immunized with CpG ODN adjuvant showed lgG1 and lgG2a readouts significantly higher than back- ground (negative control G1 = PBS only).

Conclusions

There was no significant induction of antigen specific lgG1 or lgG2a response in either GTU® or gtGTU® adjuvant groups. Thus, Th1 vs Th2 bias cannot be calculated by this data. EExceptionally high lgG1 response for just one animal in G4 is most likely caused by inflammatory reaction, for example G4 outlier may be not against OVA but BSA used as blocking reagent, or by boosting of preexisting response to OVA generated already before the start of current study (see figure 48). This has been observed previously. EXAMPLE 8

Effect ofDNA vector GTU® on adaptive immune responses

Background:

DIMA can be highly immunogenic and therapies based on DMA should prevent the de- velopment of adaptive immune responses for long term efficacy.

Preliminary results:

We found that intraperitoneal immunization of BALB/c mice with ovalbumin (OVA) plus gtGTU® or GTU® did not induce OVA-specific lgG1 or lgG2a antibodies as compared with immunization with OVA plus CpG ODN. This results suggest that the DMA vector GTU® does not induce humoral responses in mice.

Aim:

Our goal is to determine the cellular immune responses triggered by gtGTU® or GTU® to fully characterize the immunogenic activity of these DMA vectors.

Methods:

We will sensitize mice with OVA + gtGTU® or GTU® intraperitoneal on days 0, 7, 14, and 21. On day 22, we will stimulate splenocytes ex vivo with different concentrations of OVA for 3 days and collect the supernatant (soluble portion). Inflammatory cytokines present in the culture supernatants such as IFN-g, IL-1b, TNF-a, and IL-17A will be quantified by ELISA. Negative control mice will receive PBS or OVA alone during i.p. sensitization and the positive control group will be immunized with OVA + CpG ODN. Spleens from all groups will be stimulated with same concentrations of OVA.

Conclusion:

We hypothesize that i.p. immunization with the DMA vectors gtGTU® or GTU® will not induce the production of inflammatory cytokines by splenocytes as compared to CpG ODN immunization. We will conclude that vectors gtGTU® and GTU®- do not induce long-term adaptive immune responses and, therefore, are not immunogenic and safe to be further tested.

Reference listing

- Ustav, M. and Stenlund, A., EMBO J 10 (1991) 449 - 57;

- Ustav, M., et al., EMBO J 10 (1991) 4321 - 4329;

- Ustav, E. , et al. , Proc Natl Acad Sci USA 90 (1993) 898 - 902

- Ustav, M., et al., EMBO J 10 (1991) 4321 - 4329],

- HPV1a [Gopalakrishnan, V. and Khan, S,, supra],

- HPV11 [Russell, J., Botchan, M., J Virol 69 (1995) 651 -660],

~ HPV18 [Sverdrup, F. and Khan, S., J Virol 69 (1995) 1319 - 1323:

- Sverdrup, F. and Khan, S., J Virol 68 (1994) 505-509

- Ustav, M., et al., EMBO J 10 (1991) 4321 - 4329;

- Holt, S., et al., J Virol 68 (1994) 1094 - 1102;

- Holt, S. and Wilson, V. , J Virol 69 (1995) 6525 - 3652;

- Sedman, T., et al. J Virol 71 (1997) 2887 - 2996

- Ustav, E., et al., Proc Natl Acad Sci U S A. 1993 Feb 1 ;90(3):898-902

- Lusky, M., et al., Proc Natl Acad Sci USA 91 (1994) 8895-8899

- Lambert, P., et al., Annu Rev Genet 22 (1988) 235 - 258

- Androphy, E., et al., Nature 325 (1987) 70 - 739

- Howley, P. M., Papillomavirinae: the viruses and their replication, in Virology, Fields, B. C, Knipe, D. M., Howley, P. M., Eds., Philadelphia: Lippincott-Raven Publishers, 1996. 2. edition, p. 2045 - 2076

- Dostatni, N., et al., EMBO J 7 (1988) 3807 - 3816;

- Haugen, T., et al. EMBO J 7 (1988) 4245 - 4253;

- McBride, A , et al , EMBO J 7 (1988) 533 - 539;

- McBride, A., et al., Proc Natl Acad Sci USA 86 (1989) 510-514

- Hegde, R., et al., Nature 359 (1992) 505 - 512;

- Hegde, R., J Nucl Med 36(6 Suppl) (1995) 25S - 27S

- Abroi, A., et al., J Virol 70 (1996) 6169 - 6179;

- Brokaw, J., et al., J Virol 71 (1996) 23 - 29;

- Grossel, M., et al., J Virol 70 (1996) 7264 - 7269;

- Ferguson, M. and Botchan, M., J Virol 70 (1996) 4193-4199)

- Harris, S., and Botchan, M R., Science 284 (1999) 1673-1677

- Antson, A. et al., Nature 403 (2000) 805-809

- Antson, A., et al., supra

- lives, I., et al., J Virol. 73 (1999) 4404-4412

- WO02/090558

- Li et al..Genes Dev 3(4) (1989) 510-526

- Rawlins et al., Cell 42(3) (1985) 859-868;

- Reisman et al., Mol Cell Biol 5(8) (1985) 1822-1832;

- Lupton and Levine, Mol Cell Biol 5(10) (1985) 2533-2542 Goldspiel et al., Clinical Pharmacy 12 (1993) 488-505;

Wu and Wu, Biotherapy 3 (1991) 87-95;

Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993) 573-596;

Mulligan, Science 260 (1993) 926-932;

Morgan and Anderson, Ann. Rev. Biochem. 62 (1993) 191-217; May, TIBTECH 1 , l(5) (1993)155-215.

Ausubel ef a/, (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);

Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990)

WO90/11364

U.S. Patent No. 5,093,246

Langer, Science 249 (1990)1527-1533

Remington's Pharmaceutical Sciences" by E.W. Martin