OTHER DISEASES

RELATED APPLICATIONS

[ 0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/913,994, filed on October 11, 2019, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 7, 2020, is named 0371_0007W01_SL.txt and is 8,832 bytes in size.

BACKGROUND OF THE INVENTION

[ 0003] Various methods exist to deliver genetic material to cells for the expression of genetically encoded products for the treatment of genetic disorders and disease. Approaches using modified viruses show the promise and limitation of these strategies. While viruses are adept at entering cells and expressing the payloads they carry, the approaches suffer from host responses including antibody responses to viral capsid proteins, induction of cell death due to cytoplasmic host defenses that sense foreign nucleic acids and inactivation or loss of expression of the transgene payload. Added to these problems are difficulties in the manufacturability and quality assurance of the viral therapeutic.

[ 0004 ] Recent advances based on naked DNA therapeutics and closed end Adenovirus Associated Viral (ceDNA) constructs [1] improve both the ease of manufacture and quality control while alleviating problems due to capsid immunogenicity and poor transgene expression [2], The novel constructs are usually not self-repli eating, although they are often retained as episomes, limiting the effectiveness of the approach in situations the target cells are proliferating, as in tumors, due to transgene loss as cells divide.

[ 0005] Small single-stranded DNA viruses [3] also offer opportunities for building novel gene therapy products. Constructs derived from small viruses find use as vaccines to produce immune responses against desired antigens in pigs [4] and in plants for the expression of transgenes. This latter approach based on Geminivirus involves a two-step process. First a sequence containing the transgene is inserted into plasmid for Agrobacterium-mediated inoculation. The transgene is flanked by a direct repeat of the gemini virus origin of replication that recombine after entry into the host cell to reconstruct an infectious virus [5], Further amplification by the replication protein rep followed by packaging by a coat protein then allows spread of the transgene to adjacent cells. A similar approach has yet to find application in animals.

[0006] The therapeutic use of single stranded DNA viruses in humans has not been reported. In general, these classes of virus encode a rep protein that acts on a small element 41-300 nucleotides long (containing an origin of replication and the rep binding sequence, Ori-H) that engages the host machinery to ensure their replication. The viruses are remarkable in that the rep protein is able to induce their proliferation in both prokaryotes and eukaryotic cells across a wide range of species [6-8],

[ 0007 ] One example of an animal single stranded DNA virus is the porcine circovirus (PCV), which can proliferate in human cells without producing any known human disease. The potential safety of these viruses in gene therapy applications is evidenced by the lack of adverse effects following the widespread and unintentional exposure of human subjects to the intact virus when, unknown to the manufacturers involved, there was PCV contamination of the cell lines used in vaccine production [9],

SUMMARY OF THE INVENTION

[ 0008 ] The present invention describes use of plasmids for the production of circular DNA elements (CDE) that express a payload (e.g., one, or more genetic sequences such as one, or more therapeutic gene(s) suitable for expression in the claimed plasmids) for the treatment (alleviation of symptoms, or complete abrogation of) of human genetic disorders and other diseases. Also described herein are methods for the manufacture of CDE ex-vivo, either in prokaryoti c or eukaryotic systems, or in vivo. In the process, other elements of the gene delivery system such as antibody resistance genes are removed. The methods use the rep protein and the Ori-H derived from Porcine Circoviruses (PCV), although proteins and sequences from other viruses of the single-stranded DNA virus family could equivalently produce the same outcomes [10],

[ 0009] More specifically, described herein are methods for delivering genetic payloads such as therapeutic nucleic acid sequences/genes/proteins to targeted/specific cells for therapeutic treatment of a subject such as in gene therapy. The methods of the present invention comprise contacting a specific/targeted cell ex vivo or in vivo with an agent (i.e., the genetic constructs/plasmid constructs described herein) wherein the agent causes the production of (results in the production/expression of) circular DNA elements (CDE) with a desired payload, The agents/construct of the present invention are capable of (suitable for) amplification by rep and rep ' proteins, either singly or in combination.

[ooio] Also described herein are agents/ genetic constructs comprising any one of SEQ ID NOs:l through 7 and/or 9 through 13, wherein expression of the construct causes (results in) the production/expression of circular DNA elements (CDE) with a desired payload (e.g., therapeutic protein) and capable of amplification by rep and rep ' proteins, either singly or in combination.

[ooii] The methods described herein rely on a copy -release replication of the CDE [11] and do not depend on recombination between Ori-H used in the plant gene expression systems. While both animal and plant constructs have a direct repeat of the Ori-H that flanks the payload, the design differs. With plants, both flanking Ori-H are identical. In the animal system described here, the left-hand Ori-H is mutated to ensure that only the payload is amplified by the copy-release replication mechanism [7, 12], This system results in retention of the payload and loss of other vector sequences. In addition, the animal system does not infect neighboring cells, distinguishing it from the plant approach.

[ 0012 ] One feature of the rep protein important to the methods described is that it induces replication in both prokaryotic and eukaryotic systems, although the later also requires rep\ a spliced form of the rep protein called [7, 8], The constructs described here will work in both host types to produce either the vector containing the CDE payload or just the CDE alone.

[ 0013] Copy-release replication of the CDE containing constructs occurs in human cells with co-expression of rep protein increasing the rate. Efficiency of replication drops off when the size of plasmid is larger than 5.2 kilobases in vectors containing only one Ori-H [13], with the other sequence mostly consisting of vector elements that are removed by the methods described here.

[0014] One feature is that replication of the ssDNA virus occurs only in proli ferating host cells, as the rep protein can only initiate replication when the host cell DNA polymerases are active. This property allows targeting of therapeutics in ssDNA vectors to actively dividing cells such as tumor cells, or to normal (non-tumor)cells such as immune cells driven to proliferate during an antigen-specific immune response. This dependence becomes absolute with the removal of other sequences encoded by the ssDNA viruses that induce host cell proliferation, preventing any amplification in non-dividing normal cells.

[ 0015] One feature of the present invention is the use of tissue specific enhancers and promoters in the CDE resulting in expression of the payload specific for (i.e., targeted to) a particular target tissue or cell type. One example is the use of the hTERT enhancer for dividing tumor cells and a B-cell specific enhancer for use in dividing B-Cells such as that for CD20 (cluster of differentiation antigen 20, also known as membrane spanning 4- domains Al).

[0016] A further refinement of the method determines the extent of self-replication of the circular DNA produced that depends on the placement of the REP gene that encodes the rep and rep ' proteins within the production vector. If it is present between the two Ori-H, then it will allow the CDE to self-replicate as it will contain the sequence encoding the rep protein. If placed outside the Ori-H, then CDE replication will depend on the the rate of loss of rep protein through cellular process and on whether other DNA elements persist in the cell to maintain rep production.

[ 0017 ] The method covers those application where replication is through a copy-release replication mechanism and makes no claims to mechanisms involving rolling circle amplification or recombination dependent replication. The method invol ves mutations of the Ori-H to allow selective amplification of the CDE and to regulate the copy number of the CDE’s produced.

[0018] Another refinement of the method is to alter the number of rep binding sites in the Ori-H of the CDE. The Ori-H is a type of auto-regulatory iteron where rep acts both to initiate replication and to inhibit it, depending on how much rep protein is present in a cell and the number of sites that bind it [14], By varying the number of rep binding sites, it is possible to determine the copy number of CDE produced within a host. More binding sites in general leads to lower copy number by either making less rep protein available to stimulate replication or through the formation of complexes that sequester the CDE Ori-H sites, thus making them unavailable to the replication machinery [14],

[ 0019] Another refinement described herein results in preventing translation of transcripts from the REP gene that originate from the opposite strand to that producing the rep protein [15], These transcripts can produce apoptosis of the host cells and stimulate other DNA damage responses. Mutations to rep codons in the third position remove methionine initiator codons encoded on the complementary strand and prevent translation of any anti-sense RNAs produced.

[0020] Another refinement of the methods described herein results in targeting the gene therapeutic constructs to particular tissues or cells using nanoparticles, proteins, peptides, aptamers, lipids, carbohydrates or small molecules that promote uptake by, for example, tumor cells that over-express a receptor protein on their cell surface.

[0021] Another refinement of the methods described herein is to target the gene therapeutic constructs in larger vectors that permit CDE amplification in a recipient cell ex vivo for the purposes of manufacture of the CDE gene therapeutic for in vivo delivery to another cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. Of the drawings:

[ 0023] FIG. 1. A. A schema showing a production plasmid. The CDE payload is present in a production plasmid and is bounded by the Ori-H elements. The left-hand Ori-H element is mutant and only supports initiation but not termination of CDE replication. The right-handed copy supports both initi ation and proliferation of the C DE and is incorporated in the circular DNA produced. Examples of CDE cargoes are present in Figure 2. The production vector also contains an original of replication that is specific for the host used in production of the vector and contains an element that allows for selection of recombinant vectors in that host. B. In the presence of the rep protein, the C DE undergoes selective amplification by the copy-release replication mechanism and forms the circular DNA therapeutic. The rep protein can originate from the CDE, the plasmid production vector, from another plasmid, from a REP gene copy integrated into the host genome or as a protein delivered to the host from external sources. The multiple copies of the CDE are to represent its amplification. C. An illustration of the wildtype Ori-H indicating its stem, the loop containing the conserved origin of replication (boxed) and the 6 basepair direct repeat elements sequences H1, H2, H3 and H4 (SEQ ID NO: 14) [15].

[ 0024 ] FIG. 2. Examples of different schemes for encoding the CDE payload and the rep protein in the production vector. In A and B the rep protein is outside the CDE while in C and D it is part of the CDE. The composition of each element is given in the sequence listing. The ‘*’ indicates a mutant form or Ori-H.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[ 0025] The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0026] The publications discussed throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[ 0027 ] As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles "a", "an" and "the" are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods, and materials are described herein.

[ 0029] General texts, which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152, (Academic Press, Inc., San Diego, Calif.) ("Berger"); Sambrook et al., Molecular Cloning— A Laboratory Manual, 2d ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) ("Ausubel"). Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Q.beta.-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), e.g., for the production of the homologous nucleic acids of the disclosure are found in Berger, Sambrook, and Ausubel, as well as in Mullis et al. (1987) U.S. Pat. No. 4,683,202; Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press Inc. San Diego, Calif.) ("Innis"); Amheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Nafl. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem 35: 1826; Landegren et al. (1988) Science 241: 1077- 1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4:560; Barringer et al. (1990) Gene 89:117; and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references cited therein, in which PCR amplicons of up to 40 kb are generated.

[ 0030] The terms "vector", "vector construct" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology, gene synthesis or other cloning technology. A common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.

[0031] The term “single-stranded circular DNA virus” refers to those taxons described by Zhao et al. [16], regardless of the host genus.

[ 0032 ] The term PCV refers to porcine circovirus, regardless of strain. The taxonomy ID isNCBI:txid46221 (www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=46221).

The reference sequence is given by NC 013774.

(https://www.ncbi.nlm.nih.gov/nuccore/NC_013774) and by NC_001792.2 (https://www.ncbi.nlm.nih.gOv/nuccore/NC_001792.2) accessed on 10.05.2019

[ 0033] The terms “rep” and “rep ' “ refers to the proteins used by single-stranded circular DNA viruses to initiate their replication as described by Zhao et al. [16], regardless of the underlying mechanism and including those described by Preiss et al. [17], Both “rep” and

“ rep ' '' are products of the REP gene.

[ 0034 ] In one embodiment, the vector is a replication competent vector capable of infecting only replicating tumor cells with particular mutations. In one embodiment, a replication competent vector comprises an internal ribosomal entry site (IRES), or viral sequences for bicistronic expression, 5' to the heterologous polynucleotide encoding, e.g., a cytosine deaminase, miRNA, siRNA, cytokine, receptor, antibody or the like. When the heterologous polynucleotide encodes a non-translated RNA such as siRNA, miRNA or RNAi then no IRES is necessary, but may be included for another translated gene, and any kind of viral construct can be used. In one embodiment, the polynucleotide is 3' to an ENV polynucleotide of a retroviral vector. In one embodiment, the viral vector is a retroviral vector capable of infecting targeted tumor cells multiple times (5 or more per diploid cell).

[ 0035] The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be "expressed" by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.

[ 0036] The terms “gene editing” or “gene editing techniques” as described herein can include RNA-mediated interference (referred to herein as RNAi, or interfering RNA molecules), or Short Hairpin KNA (shRNA) or CRISPR-Cas9 and TALEN. See e.g., Agrawal. N. et al., Microbiol Mol Biol Rev. 2003 Dec; 67(4): 657-685; Moore, C.B., et al. Methods Mol Biol. 2010; 629: 141-158; Doudna, J.A. and Charpentier, E. Science vo. 346, 28 Nov. 2014; Sander, J.D. and Joung, K. Nature Biotech 32, 347-355 (2014); U.S. Pat 8,697,359; Nemudryo, A. A. ACTA Naturae vol. 6, No. 3(22)2014. Anti-sense RNA can also be used. (Gleave, M. and Monia, B., Nature Reviews Cancer 5, 468-479 (June 2005)). The term “gene therapy” generally means a method of therapy wherein a desired gene/genetic sequence is inserted into a cell or tissue (along with other sequences necessary for the expression of the specific gene). See, for example, genetherapynet.com for description of gene therapy techniques.

[ 0037 ] The term "subject" as used herein can include a human subject for medical purposes, such as for the treatment of an existing disease, disorder, condition or the prophylactic treatment for preventing the onset of a disease, disorder, or condition or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; capiines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, guinea pigs, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a "subject" can include a patient afflicted with or suspected of being afflicted with a disease, disorder, or condition. Thus, the terms "subject" and "patient" are used interchangeably herein. Subjects also include animal disease models (e.g., rats or mice used in experiments, and the like).

[ 0038 ] The term “CDE” is an abbreviation of a circular DNA element. It applies to the sequence in a production vector used to manufacture a circular DNA possessing a single Ori-H, where the production vector also contains a second Ori-H to define the 5' end of the CDE sequence (Fug. 1 A). The term CDE also applies to the product manufactured from the production vector that consists of circular DNA containing a payload and a single Ori-H (Fig. IB)

[0039] The term CMV stands for human cytomegalovirus.

[0040] The term hTERT stands for the human telomerase.

[0041] The term RU5' stands for R segment and a part of the U5 sequence (R-U50) of the HTLV type 1 long terminal repeat.

[0042] The term WPRE stands for the woodchuck hepatiti s virus posttranscriptional regulatory element.

[ 0043] The term “production vector” refers to the construct shown in Fig. 1 A where the CDE is embedded in a larger circular DNA (or an RNA template) that has a host-specific origin of replication and selection marker and two copies of the Ori-H that direct production of the CDE by a copy-release mechanism.

[ 0044 ] The term "cancer" or “tumor” includes, but is not limited to, solid tumors and blood borne tumors. These terms i nclude diseases of the skin, tissues, organ s, bone, cartilage, blood and vessels. These terms further encompasses primary and metastatic cancers.

[ 0045] The term “copy-release replication” means to copy one strand of a nuclei c acid template, release the product, then synthesize the complementary strand in a second step that involves a new replication complex, either with the same components as the first replication complex or using a different set of enzymes. It does not require a rolling circle mechanism that replicates an entire circular DNA template. Instead copy-release amplifies only the DNA segment between the two Ori-H is amplified. In the case of single-stranded DNA virus, rep proteins initiate replication of the first strand by host DNA polymerases. Synthesis of the complementary strand also involves host polymerases and occurs following the release of the copy from the viral template. In the case of a circular ssDNA virus, replication of DNA both begins and ends at an Ori-H and leads to the production of a double-stranded circular DNA from which further single-stranded copies can be made.

[0046] The term “rolling circle replication” refer to replication that proceeds around a circular DNA template, returning to the start position, then repeating the cycle. In the process, all the template is copied multiple times. [ 0047 ] The term “recombination-dependent replication” refers to the process where recombination between two Ori-H results in a closed, circular, covalently linked DNA to create a template for replication .

[ 0048 ] The term “origin of replication” of a circular ssDNA virus refers to a conserved nonanucleotide sequence (where “N” is any nucleotide and J, indicates a cleavage site [3, 15]). The sequence is in a loop bounded by a double-stranded stem. Attached to the stem is a variable number of rep protein binding sites, designated as H. Together, the origin of replication loop and stem, plus the repeat elements form the Ori-H. In PCV, H represents a direct repeat of at least one 8 basepair sequence and is 11 basepairs in the wildtype virus [12, 18], In the case of PCV, the conserved sequence at the origin of replication is NAGTATTAC (Fig. 1C). The sequence of the stem may be natural or contain variations to alter the energetics of stem formation. The rep binding sites may be those found in nature or selected to optimize binding to a particular rep variable, using a screening method known to those of skill where different stem configurations and rep variants are tested to find combinations with a prescribed rate of replication for the single-stranded virus.

[ 0049] The methods described herein use vectors that contain two Ori-H copies and the payload in the following order (Fig. 2). First a mutated form of the PCV Ori-H that allows initiation of replication but not termination of the CDE [12], then a payload of interest and then a wildtype Ori-H. In addition, the vector can contain other sequences outside the CDE that allow for replication of the entire construct independently of rep protein, in either prokaryotic or eukaryotic cells, plus other sequences that allow selection of recombinant plasmids (Fig. 1 A). The additional sequences enable construction of a vector for producing multiple copies of the CDE.

[0050] A sequence encoding the rep protein may be placed in the vector, either in association with the payload between the two Ori-H sequences to ensure sufficient levels of rep and rep' so regulate production of the CDE from the production vector. High levels of the rep and rep ' will inhibit production of further CDE once they reach a certain copy number.

[0051] For vectors that lack a gene encoding the rep protein, expression of the rep protein is from a separate vector, from a transgene present in the host cell genome, or from a mRNA or as a protein introduced into the cell by transfection, electroporation, sonication, ballistic projectile, nanoparticles or any other means. [ 0052 ] Likewise in eukaryotic cells, the rep ' protein arises from a splice isoform of the REP gene transcript, or from a separate construct, either on the same vector, on a separate vector or from a transgene in the host cell genome. The rep ' protein can also derive from a rhRNA or a recombinant protein introduced directly into the cell by transfection, electroporation, sonication, ballistic projectile or any other means including nanoparticles and in association with cell-penetrating molecules.

[ 0053] The Ori-H element prototype (Fig. 1C) contains a conserved loop nonamer (NAGTATTAC) where cleavage at site indicated by the arrow is essential to initiation and and termination of replication. The double-stranded stem to which it is attached needs to be at least 8 basepairs long, although its total length nucleotide sequence can vary provided all bases are paired [19],

[ 0054 ] In one embodiment, a production vector embeds the CDE, with an additional mutant Ori-H at its 5' end, and other sequences that allow for the replication and selection of the entire vector in a particular host. The production vector for the CDE may be used directly as a therapeutic by its introduction into the targeted tissue. In another embodiment, the production vector is used in its preferred host to amplify the CDE, which is then purified and used as the therapeutic. In both cases, the CDE can be amplified in the targeted tissue by producing rep in that tissue. The CDE can also be used without a system for producing rep in the host system.

[ 0055] Each CDE DNA sequence to be expressed as RNA is associated with other DNA sequences that enhance expression and or encode elements that regulate the stability of the RNAs produced (Fig. 2). These regulatory sequences are well-known to those skilled in the art and examples are provided in the sequences listed.

[ 0056] These methods are useful for all proteins where expression is necessary for a therapeutic effect.

[ 0057 ] The examples include complement-derived proteins such as C3, C3b, iC3b, C3c, C3d, C4, C4d, C5 , C3aRl, C5aRl, C5aR2, C1R, CTRL, CR2, C1QBP, CD46, CD55, CD59, or LAIR1 in tumors in order to provoke an antigen-specific immune response against the tumor.

[ 0058 ] The term “antigen” is defined as any molecule that a T-Cell or B-Cell receptor has specificity for, or any molecule targeted by Natural Killer Cells or other Innate Cells that specifically targets their effector function such as cytotoxic killing of cells, release of growth factors, lymphokines or cytokines. (Microbiology and Immunology On-line, Edited by Richard Hunt, PhD; www.microbiologybook.org/mayer/antigens2000.htm)

[0059] The term “CAR” refers to any chimeric antigen receptor introduced into immune cells for therapeutic purposes using a gene therapy approach, like the one described here

[20].

[0060] The term “CSP” refers to complement split products produced by proteolysis of complement proteins [21], For the purposes of this invention, it specifically refers to proteolytic fragments produced from complement components C3 and C4 that activate immune responses (“actCSP”) or inhibit them (“inhCSP”) that is administered using the CDE described in this method.

[0061] The methods and compositions of the present invention may be used to treat any type cancerous tumor or cancer cells. Such tumor s/cancers may be located anywhere in the body, including without limitation in a tissue selected from brain, colon, urogenital, lung, renal, prostate, pancreas, liver, esophagus, stomach, hematopoietic, breast, thymus, testis, ovarian, skin, bone marrow and/or uterine tissue. Cancers that may treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; ley dig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; e wing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. [ 0062 ] A “therapeutically effective” amount as used herein refers to an amount sufficient to have the desired biological effect (for example, an amount sufficient to express the GPI- anchored CSPs to produce the desired effect on the underlying disease state (for example, an amount sufficient to inhibit tumor growth in a subject, produce an immune response to an antigen or to inhibit autoimmune disease) in at least a sub-population of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment. Determination of therapeutically effective amounts of the agents used in this invention, can be readily made by one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. The amounts/dosages may be varied depending upon the requirements of the subject in the judgment of the treating clinician; the severity of the condition being treated and the particular composition being employed. In determining the therapeutically effecti ve amount, a number of factors are considered by the treating clinician, including, but not limited to: the specific disease state; pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species being treated; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the particular agent administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment (i.e., the interaction of the agent with other co-administered agents); and other relevant circumstances.

[ 0063] For example, as described herein, the amino acid sequence of the iC3b, C3c, C3d, C4b and C4d proteins can be truncated/mutated/altered to produce biologically active peptides or variants. Such peptides derived from the iC3b,C3c, C3d, C4b and C4d protein can be synthesized, or otherwise produced and evaluated for their biological activity. Biological activity can include binding of iC3b ,C3c, C3d, C4b and C4d peptides to MHC, or change of sites of proteolysis by proteases such as metalloproteinases, or include sites of proteolysis that result in removal of the GPI tag so that it is released into the extracellular environment. Mutations can specifically increase MHC binding to increase immunomodulation.

[ 0064 ] In certain embodiments, the agents described for use in this invention can be combined with other pharmacologically active compounds ("additional active agents") or peptide antigens (“antigens”) known in the art according to the methods and compositions provided herein. Additional active agents can be large molecules (e.g., proteins, lipids, carbohydrates) or small molecules (e.g., synthetic inorganic, organometallic, or organic molecules). In one embodiment, additional active agents independently or synergistically help to treat cancer.

[ 0065] For example, certain additional active agents are anti -cancer chemotherapeutic agents. The term chemotherapeutic agent includes, without limitation, platinum-based agents, such as carboplatin and cisplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents, such as carmustine (BCNU) and other alkylating agents; antimetabolites, such as methotrexate; purine analog antimetabolites; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine; hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as taxanes (e.g., docetaxel and paclitaxel), aldesleukin, interleukin-2, etoposide (VP- 16), interferon alfa, and tretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin, dactinomycin, daunorubicin, doxorubicin, and mitomycin; and vinca alkaloid natural antineoplastics, such as vinblastine and vincristine or agents targeted at specific mutations within tumor cells.

[ 0066] Further, the following drugs may also be used in combination with an antineoplastic agent, even if not considered antineoplastic agents themselves: dactinomycin; daunorubicin HC1; docetaxel; doxorubicin HC1; epoetin alfa; etoposide (VP- 16); ganciclovir sodium; gentamicin sulfate; interferon alfa; leuprolide acetate; meperidine HC1; methadone HC1; ranitidine HC1; vinblastin sulfate; and zidovudine (AZT). For example, fluorouracil has recently been formulated in conjunction with epinephrine and bovine collagen to form a particularly effective combination.

[ 0067 ] Still further, the following li sting of amino acids, peptides, polypeptides, proteins, polysaccharides, and other large molecules may also be used in conjunction with the invention: checkpoint inhibitors that target for example, PD-1 and CTLA-4, interleukins 1 through 37, including mutants and analogues; interferons or cytokines, such as interferons .alpha., .beta., and .gamma.; hormones, such as luteinizing hormone releasing hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors, such as transforming growth factor-.beta. (TGF-.beta.), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homologous factor (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF); tumor necrosis factor-. alpha. & .beta. (TNF-. alpha. & .beta.); invasion inhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-, alpha. - 1 ; .gamma. -globulin; superoxide dismutase (SOD); complement factors; anti-angiogenesis factors; antigenic materials; and pro-drugs.

[0068] Chemotherapeutic agents for use with the compositions and methods of treatment described herein include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); ciyptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBl-TMl); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; acegl atone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfonnithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difl uoromethylomithi ne (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[ 0069] The compositions and methods of the invention can comprise or include the use of other biologically active substances, including therapeutic drugs or pro-drugs, for example, other chemotherapeutic agents or antigens useful for cancer vaccine applications. Various form s of the chemotherapeutic agents and/or additi onal active agents may be used. These include, without limitation, such forms as uncharged molecules, molecular complexes, salts, ethers, esters, amides, and the like, which are biologically active.

[ 0070] The agents and substances described herein can be delivered to the subject in a pharmaceutically suitable, or acceptable or biologically compatible carrier. The terms “pharmaceutically suitable/acceptable” or “biologically compatible” mean suitable for pharmaceutical use (for example, sufficient safety margin and if appropriate, sufficient efficacy for the stated purpose), particularly as used in the compositions and methods of this invention.

[0071] The compositions described herein may be delivered by any suitable route of administration for treating the cancer, including orally, nasally, transmucosally, ocularly, rectally, intravaginally, parenterally, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra- sternal, intra-synovial, intra-hepatic, through an inhalation spray, or other modes of delivery known in the art.

[ 0072 ] The nucleic acid sequence for C3, including the CSPs iC3b, C3d and C3dg can be found e.g., in Proc. Natl. Acad. Sci. USA, vol. 82, pp. 708-712, February 1985). The term “C3d” as used herein is intended to encompass both C3d and C3dg and the term “iC3b”is used to encompass “C3c”. The nucleic acid sequence for the C3aR can be found at “C3AR1 complement C3a receptor 1 [ Homo sapiens (human) ]” Gene ID: 719, www.ncbi.nlm.nih.gov/gene, updated on 6-Aug-2017. The nucleic acid sequence for the C5a receptor can be found at “C5AR1 complement C5a receptor 1 [ Homo sapiens (human)]” Gene ID: 728, www.ncbi.nlm.nih.gov/gene, updated on 29-Aug-2017. C1R complement Clr [ Homo sapiens (human) ], Gene ID: 715, www.ncbi.nlm.nih.gov/gene , updated on 3-Sep-2017, C1RL complement Clr subcomponent like [ Homo sapiens (human) , Gene ID: 51279, www.ncbi.nlm.nih.gov/gene, updated on 3-Sep-2017, C5AR2 complement component 5a receptor 2 [ Homo sapiens (human) ],Gene ID: 27202, www.ncbi.nlm.nih.gov/gene, updated on 3-Sep-2017, C1QBP complement Clq binding protein [ Homo sapiens (human)}, Gene ID: 708, www.ncbi.nlm.nih.gov/gene, updated on 3-Sep-2017, CR2 complement C3d receptor 2 [ Homo sapiens (human) ], Gene ID: 1380, www.ncbi.nlm.nih.gov/gene, updated on 3-Sep- 2017, CD46 molecule [ Homo sapiens (human) ], Gene ID: 4179, www.ncbi.nlm.nih.gov/gene, updated on 3-Sep-2017, CD55 molecule (Cromer blood group) [ Homo sapiens (human) ], Gene ID: 1604, www.ncbi.nlm.nih.gov/gene, updated on 6-Sep- 2017, CD59 molecule (CD59 blood group) [ Homo sapiens (human) ], Gene ID: 966, www.ncbi.nlm.nih.gov/gene, updated on 3-Sep-2017 andLAIRl leukocyte associated immunoglobulin like receptor 1 [ Homo sapiens (human) ],Gene ID: 3903, www.ncbi.nlm.nih.gov/gene, updated on 3-Sep-2017. The nucleic acid sequence for the proteases cathepsin L [Homo sapiens (human)], CTSL ,Gene ID: 1514 and cathepsin S [Homo sapiens (human) ], CTSS, Gene ID: 1520, can be found atwww.ncbi.nlm.nih.gov/gene, updated on 3-Sep-2017.

[ 0073] For example, a gene editing technique to produce the C3d or C3c transcript within tumors can be used so that the protein product is targeted to the cell surface membrane as described in this invention (see e.g., US Patent 8,697, 359 for a description of CRISPR techniques). Delivery of CRISPR/CAS9 with a sgRNAs to C3 (excluding the C3d sequence) and the nucleic acid sequences for C3d or C3d derived peptides, to a tumor cell can be provided by use of a viral vector. Delivery of CRISPR/CAS9 with a sgRNAs to C3 (excluding the C3c sequence) and the nucleic acid sequences for C3c or C3c derived peptides to a tumor cell along with other sequences necessary for targeting of the CSP transcripts that are introduced into cleavage sites during the process of repair, can be provi ded by use of a viral vector. A number of viral vectors have been used in humans and these can be used to transduce the genetic material in different cell types. Those of skill in the art know such methods. Means to target the vectors for specific delivery of the constructs to the tumor cells of interest are also known to those of skill. For example, genetically engineered vectors exist where the capsid is modified to contain ligands for receptors that facilitate viral entry onto a particular cell type. An example is given in Figure 1. This construct also includes a reporter gene that allows efficiency of transduction of the virus into the tumor to be quantitated.

[ 0074 ] The above approaches permit combination with other cancer therapies including immune-modulators such as checkpoint inhibitor ligands for PD-1 CTLA-4, ICOS, 0X40; reagents against C3a and C5a receptors; lymphokines, cytokines and their receptors and strategies designed to increase major and minor histocompatibility antigens. Additionally, the methods of the present invention can be combined with other standard cancer therapies such as radiotherapy and chemotherapy.

Sequence Examples

1. RU5' sequence (269 bp: Accession No. J02029 (374-642) (SEQ ID NO: 1)

2. hTERT Enhancer sequence (189 bp: Accession No. DQ264729 (1618-1806)) (SEQ ID NO:2) i . ctt

3. CMV Promoter sequence: (171 bp) (includes ribosome binding site plus start codon (underlined)) (SEQ ID NO:3)

4. CMV Enhancer sequence (304 bp) (SEQ ID NO:4)

5. WRE poly-A sequence (448 bp) (SEQ ID NO:5)

6. 2A Sequence (66 bp) (SEQ ID NO:6)

7. PC VI Rep Sequence (939 bp) (with mutations to remove initiator methionine from complementary strand and amino acid translation)(ref NC_001792.2) (SEQ ID NOS: 7 and 8)

8. Ori-H Sequence Minimal (53 bp) (SEQ ID NO:9)

9. Ori-H Sequence Wildtype (70 bp) (SEQ ID NO: 10)

10. Ori-H Sequence Mutant with deletion of left stem sequence (59 bp) (SEQ ID NO: 11)

11. Ori-H Sequence Mutant with mutant left stem sequence shown in lower case (70 bp) (SEQ ID NO: 12)

12. Ori-H Sequence Mutant with mutant left stem sequence with rep promoter (70 bp) (SEQ ID NO: 13

References

1 Li, L., etal. (2013) Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer. PLoS One 8, e69879

2 Hardee, C.L., etal. (2017) Advances in Non-Viral DNA Vectors for Gene Therapy. Genes (Base l) 8

3 Rosario, K„ etal. (2012) A field guide to eukaryotic circular single-stranded DNA viruses: insights gained from metagenomics. Arch Virol 157, 1851-1871

4 Pineyro, P.E., et al. (2016) Evaluation of the use of non-pathogenic porcine circovirus type 1 as a vaccine delivery virus vector to express antigenic epitopes of porcine reproductive and respiratory syndrome virus. Virus Res 213, 100-108 5 Yang, Q.-y., et al. (2017) Gemini viruses and their application in biotechnology. Journal of Integrative Agriculture 16, 2761-2771

6 Kammann, M., etal. (1991) Gemini virus-based shuttle vectors capable of replication in Escherichia coli and monocoty 1 edonous plant cells. Gene 104, 247-252

7 Cheung, A.K. (2006) Rolling-circle replication of an animal circovirus genome in a theta- replicating bacterial plasmid in Escherichia coli. J Virol 80, 8686-8694

8 Steinfeldt, T., etal. (2006) Demonstration of nicking/joining activity at the origin of DNA replication associated with the rep and rep' proteins of porcine circovirus type 1. J Virol 80, 6225-6234

9 Denner, J. and Mankertz, A. (2017) Porcine Circoviruses and Xenotransplantation. Viruses

9

10 Shulman, L.M. and Davidson, I. (2017) Viruses with Circular Single-Stranded DNA Genomes Are Everywhere! Annu Rev Virol 4, 159-180

11 Cheung, A.K. (2015) Specific functions of the Rep and Rep proteins of porcine circovirus during copy-release and rolling-circle DNA replication. Virology 481, 43-50

12 Cheung, A.K. (2004) Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model. J Virol 78, 9016-9029

13 Faurez, F., etal. (2010) Replication efficiency of rolling-circle replicon-based plasmids derived from porcine circovirus 2 in eukaryotic cells. J Virol Methods 165, 27-35

14 Paulsson, J. and Chattoraj, D.K. (2006) Origin inactivation in bacterial DNA replication control. Mol Microbiol 61, 9-15

15 Cheung, A.K. (2012) Porcine circovirus: Transcription and DNA replication. Virus Res 164, 46-53

16 Zhao, L., et al. (2019) Eukaryotic Circular Rep-Encoding Single-Stranded DNA (CRESS DNA) Viruses: Ubiquitous Viruses With Small Genomes and a Diverse Host Range. Adv Virus Res 103, 71-133

17 Preiss, W. and Jeske, H. (2003) Multitasking in replication is common among gemini viruses. J Virol 77, 2972-2980

18 Cheung, A.K. (2005) Mutational analysis of the direct tandem repeat sequences at the origin of DNA replication of porcine circovirus type 1. Virology 339, 192-199

19 Cheung, A.K. (2007) A stem-loop structure, sequence non-specific, at the origin of DNA replication of porcine circovirus is essential for termination but not for initiation of rolling- circle DNA replication. Virology 363, 229-235

20 Maldini, C.R., et al. (2018) CAR T cells for infection, autoimmunity and allotransplantation. Nat Rev Immunol 18, 605-616

21 Hajishengallis, G, etal. (2017) Novel mechanisms and functions of complement. Nat Immunol 18, 1288-1298