Non-integrating viral delivery system and methods of use thereof

Abstract

To provide non-integrating viral delivery systems, and to provide methods of use thereof.SOLUTION: The present invention relates generally to non-integrating viral delivery system and to methods of using the same. The viral delivery system includes a viral carrier, a heterologous viral episomal origin of replication, a sequence encoding at least one initiator protein specific for the heterologous viral episomal origin of replication, and at least one gene, shRNA, siRNA, miRNA, or other gene-silencing RNA of interest. In certain embodiments, the disclosed system can be used for gene therapy.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority and Incorporation by Reference This application claims priority to U.S. Provisional Application No. 62 / 173,748, filed June 10, 2015, entitled "Non-Integrating Viral Delivery System and Methods of Use Thereof," which is incorporated by reference in its entirety.

[0002] Field The present invention relates generally to the field of viral vectors and systems for gene delivery and other therapeutic, diagnostic, or research uses. [Background technology]

[0003] background Viral vectors can be used to transduce genes into target cells due to the specific interaction of viral envelope-host cell receptors and the viral mechanism for gene expression.As a result, viral vectors have been used as vehicles for gene transfer into many different cell types, including whole embryos, fertilized eggs, isolated tissue samples, in situ tissue targets, and cultured cell lines.The ability to introduce and express foreign genes into cells is useful for studying gene expression and elucidating cell lineages, and providing the possibility of therapeutic interventions such as gene therapy, somatic cell reprogramming of induced pluripotent stem cells, and various types of immunotherapy.

[0004] Given the wide variety of potential genes available for gene therapy, efficient means of delivering these genes are desirable. Several viral systems, including murine retroviruses, adenoviruses, and parvoviruses (adeno-associated viruses), have been developed as therapeutic gene transfer vectors. Many factors must be considered when developing viral vectors, including expression stability and control, genome packaging capacity, and construct-dependent vector stability. Furthermore, the in vivo application of viral vectors is often limited by host immune responses to viral structural proteins and / or transduced gene products.

[0005] One approach for producing recombinant polypeptides or gene regulatory molecules, including small RNAs, is the use of stable expression systems. These systems are based on chromosomal integration of the transduced retroviral genome (or at least a portion thereof) into the host cell genome, short-term plasmid transfection, or non-integrating viral vectors with limited half-lives. The site of gene integration is generally random, and the number and proportion of genes integrated at any particular site is often unpredictable. Similarly, non-integrating plasmid or viral vectors also produce nuclear DNA, but these species typically lack the sequences necessary for DNA replication and continuous maintenance. Therefore, vectors that rely on chromosomal integration result in persistent maintenance of the recombinant gene, which may exceed the therapeutic interval, and the control of the plasmid or other non-replicating DNA is insufficient and may decay before the desired therapeutic period is completed.

[0006] An alternative to stable expression systems for gene expression is transient expression systems. Expression in transient gene expression systems is based on non-integrated plasmids, and therefore expression is typically lost as cells divide, or the plasmid vector is destroyed by endogenous nucleases. Thus, transient gene expression systems typically result in poor expression over time, traditionally requiring repeated treatments, which is generally undesirable. Summary of the Invention [Means for solving the problem]

[0007] Abstract In one embodiment, the present invention relates to a novel non-integrating viral delivery system and methods of use thereof.

[0008] In one aspect, the present invention relates to a viral delivery system comprising a viral carrier, a heterologous viral episomal origin of replication, a sequence encoding at least one initiator protein specific for the heterologous viral episomal origin of replication, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene-regulating RNA, wherein the viral carrier has a defective integrase gene and expression of the sequence encoding at least one initiator protein specific for the heterologous viral episomal origin of replication is under the control of an inducible promoter.

[0009] In some embodiments, viral carrier is lentivirus, but other viral carriers can also be suitable.In some embodiments, heterologous virus episome replication origin is derived from papillomavirus, such as bovine papillomavirus or human papillomavirus, and at least one initiator protein specific for heterologous virus episome replication origin is papillomavirus E1 and / or E2.In certain embodiments, both E1 and E2 exist in the system described herein.

[0010] Alternatively, in some embodiments, the heterologous viral episomal origin of replication is derived from either Epstein-Barr virus or a related mammalian herpesvirus, and the at least one initiator protein specific for the heterologous viral episomal origin of replication is EBNA-1 or a specific initiator protein found in each individual herpesvirus.

[0011] In another aspect, the disclosed invention relates to a pharmaceutical composition comprising the disclosed viral delivery system and a pharmaceutically acceptable carrier.

[0012] In another aspect, the disclosed invention relates to a method for treating or preventing a disease, comprising identifying a subject in need thereof and administering to the subject a therapeutically effective amount of a viral delivery system according to the present invention. The viral delivery system comprises a viral carrier, a heterologous viral episomal origin of replication, a sequence encoding at least one initiator protein specific to the heterologous viral episomal origin of replication, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene silencing RNA, wherein the viral carrier has a defective integrase gene or integration is silenced by a pharmaceutical inhibitor of the viral integrase protein, and wherein expression of the sequence encoding at least one initiator protein specific to the heterologous viral episomal origin of replication is under the control of an inducible promoter.

[0013] In some embodiments, the disease may be an infectious disease such as Ebola virus, Lassa virus, or any viral agent that can be protected against by a specific antibody, and in these embodiments, the gene of interest, shRNA, siRNA, miRNA, and / or other gene silencing RNA may encode an antibody specific for Ebola virus or Lassa virus or any other specific viral target.

[0014] In some embodiments, the disease may be an infectious disease such as Staphylococcus aureus or Escherichia coli, or other bacterial, fungal, or protozoan pathogen, where the gene of interest, shRNA, siRNA, miRNA, and / or other gene silencing RNA encodes an antibody that targets to reduce replication of the pathogen or block the activity of a toxin produced by the exogenous pathogen.

[0015] In some embodiments, the disease to be treated or prevented using the disclosed methods is a genetic disease or disorder.In some embodiments, the genetic disease is alcohol abuse, and the gene of interest, shRNA, siRNA, miRNA, and / or other gene silencing RNA encodes a brain-derived growth factor.

[0016] In another aspect, the disclosed invention relates to a method for treating nerve injury, comprising identifying a subject with nerve injury and administering to the subject a therapeutically effective amount of a viral delivery system according to the present invention. The viral delivery system comprises a viral carrier, a heterologous viral episomal origin of replication, a sequence encoding at least one initiator protein specific to the heterologous viral episomal origin of replication, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA, wherein the viral carrier has a defective integrase gene, and expression of the sequence encoding at least one initiator protein specific to the heterologous viral episomal origin of replication is under the control of an inducible promoter. In some embodiments, the gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA is a nerve growth factor.

[0017] In yet another embodiment, the disclosed invention relates to a method for enhancing wound healing, comprising identifying a subject with a wound and administering to the subject a therapeutically effective amount of a viral delivery system according to the present invention. The viral delivery system comprises a viral carrier, a heterologous viral episomal origin of replication, a sequence encoding at least one initiator protein specific to the heterologous viral episomal origin of replication, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA, wherein the viral carrier has a defective integrase gene, and expression of the sequence encoding at least one initiator protein specific to the heterologous viral episomal origin of replication is under the control of an inducible promoter. The wound may be caused, for example, by an accident, injury, or surgery. In some embodiments, the gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA is platelet-derived growth factor or vascular endothelial growth factor.

[0018] In another embodiment, the disclosed invention relates to a method for enhancing recovery from traumatic skin burns, comprising identifying a subject with a traumatic burn and administering to the subject a therapeutically effective amount of a viral delivery system according to the present invention. The viral delivery system may comprise a viral carrier, a heterologous viral episomal replication origin, a sequence encoding at least one initiator protein specific to the heterologous viral episomal replication origin, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA, wherein the viral carrier has a defective integrase gene and expression of the sequence encoding at least one initiator protein specific to the heterologous viral episomal replication origin is under the control of an inducible promoter. The skin trauma may be caused by chemical or physical (heat, ultraviolet light, or radiation) damage. In some embodiments, the gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA encodes an epidermal or keratinocyte growth factor or other promoter for epidermal cell growth and differentiation.

[0019] In another embodiment, the disclosed invention relates to a method for treating cancer or neoplastic disease, comprising identifying a subject having cancer or neoplastic disease and administering to the subject a therapeutically effective amount of a viral delivery system according to the present invention. The viral delivery system may comprise a viral carrier, a heterologous viral episomal origin of replication, a sequence encoding at least one initiator protein specific for the heterologous viral episomal origin of replication, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene silencing RNA, wherein the gene of interest, shRNA, siRNA, miRNA, and / or other gene silencing RNA encodes an antibody targeting a tumor antigen, or a polypeptide and / or regulatory RNA intended to alter the sensitivity of the tumor to immunotherapy, surgery, radiation, or chemotherapy.

[0020] In another embodiment, the disclosed invention is a system for delivering proteins and / or nucleic acids, including a system for gene deletion, modification, or re-sequencing.Current technology for modifying chromosomal genes lacks appropriate safety factors to prevent off-target effects.In the proposed invention, the gene encoding the DNA modification system of the present invention is reliably depleted after the required treatment period.

[0021] In another embodiment, the disclosed invention relates to a method for enhancing bone healing, comprising identifying a subject with a wound and administering to the subject a therapeutically effective amount of a viral delivery system according to the present invention. The viral delivery system comprises a viral carrier, a heterologous viral episomal origin of replication, a sequence encoding at least one initiator protein specific to the heterologous viral episomal origin of replication, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA, wherein the viral carrier has a defective integrase gene and expression of the sequence encoding at least one initiator protein specific to the heterologous viral episomal origin of replication is under the control of an inducible promoter. The fracture may be due to, for example, an accident, injury, trauma, or surgery, and may be a nonunion or acute fracture of a bone or a required spinal fusion. In some embodiments, the gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA encodes bone morphogenetic protein 1-4, cyclooxygenase-2, or vascular endothelial growth factor.

[0022] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the invention. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a diagram illustrating an exemplary virus within a viral vector according to an embodiment of the present invention. [Figure 2] FIG. 2 shows an exemplary vector-in-vector (VIV) embodiment (Vector 1) that also contains an E1 initiator protein. [Figure 3] FIG. 3 shows the results of transduction in 293T cells using Vector 1. [Figure 4]Figure 4 shows an exemplary vector-in-vector embodiment that also contains both E1 and E2 initiator proteins. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description Disclosed herein are non-integrative, episomally replicating viral vectors (e.g., lentiviral vectors) and methods for their use. Episomal replicating vectors such as those of the present invention may contain viral components from Papovaviridae (e.g., bovine papillomavirus, or BPV), Herpesviridae (e.g., Epstein-Barr virus, or EBV), or Hepadnaviridae (e.g., hepatitis B virus, or HBV). Episomal replicating vectors derived from these viruses may contain an origin of replication and at least one viral transactivator, such as an initiator protein such as E1 for BPV polymerase and EBNA-1 for EBV or HBV polymerase, or an adenoviral terminal binding protein. The process of episomal replication typically incorporates both the host cell's replication machinery and viral transactivators.

[0025] By using a heterologous viral origin of replication, novel vectors can be engineered with an "off" switch for the expression of viral proteins required to recognize the origin of replication. Switching off the vector's DNA replication prevents further expression of the transduced gene, and the viral vector fades over time, resulting in the transduced gene no longer being present in the host cell. The disclosed systems and methods offer many improvements over the prior art, including the ability to prevent any toxic effects resulting from overexpression or prolonged expression of the transduced gene. Eliminating the gene once DNA replication has stopped prevents subsequent undesired gene expression or knockdown. Similarly, combining the benefits of episomal replication with heterologous viral systems provides a platform that can safely and efficiently transduce genes of interest into a variety of cell types.

[0026] Papillomavirus Papillomaviruses replicate primarily episomally in mammalian cells. The action of the viral E1 protein, which functions as a DNA helicase, on the viral origin of replication (ori) drives the production of hundreds to thousands of copies per cell, depending on the differentiation state of the infected epithelial cells. Recognizing this property, several laboratories attempted to develop papillomavirus-based gene delivery systems using what have become known as "shuttle plasmids." Using bacterial origins of replication to enable DNA production in E. coli and papillomavirus oris to enable episomal replication in mammalian cells, numerous studies have been conducted to demonstrate the safety and durability of gene expression. In most cases, the oris have been derived from bovine papillomavirus.

[0027] Papillomaviruses have evolved to infect epidermal and epithelial cells. As infected cells differentiate from the basal to the luminal surface, papillomaviruses increase their DNA replication, and the copy number increases dramatically until large amounts of virus are released at the luminal surface. This makes papillomaviruses highly infectious, as evidenced by human papillomaviruses. The rapid increase in copy number is primarily due to host factors. However, this feature of papillomaviruses can be exploited for transient gene therapy to target the epidermal and epithelial surfaces.

[0028] Thus, certain features of papillomaviruses can be used to drive the expression and replication of episomal vectors, as well as to target the expression of the vectors to specific cell types.

[0029] Epstein-Barr virus (EBV) Epstein-Barr virus (EBV), also known as human herpesvirus 4, is a member of the herpesvirus family. It is one of the most common human viruses, and most people will be infected with EBV at some point in their lives.

[0030] EBV is a double-stranded DNA virus that contains approximately 85 genes and is known to infect B cells and epithelial cells. EBV is capable of both lytic and latent replication, which results in the translocation of a circularized form of the EBV genome into the host cell nucleus, where it can be replicated by host cell DNA polymerases.

[0031] EBV can replicate latently through at least three distinct pathways, each of which involves the expression of Epstein-Barr virus nuclear antigen 1 (EBNA-1), a protein that binds episomal replication origins and mediates episomal compartmentalization during host cell division. EBNA-1 plays an essential role in EBV gene regulation, replication, and episomal maintenance.

[0032] Due to its natural tropism and ability to replicate extrachromosomally, EBV is of interest as a viral vector. Such vectors are often mutated to delete EBV viral oncogenes and / or crucial lytic genes. However, the utility of EBV as a viral vector has so far been largely limited to ex vivo and research purposes.

[0033] Hepatitis B virus (HBV) Hepatitis B virus (HBV) is a member of the hepadnavirus family. It is a common human virus associated with progressive liver fibrosis, hepatitis, and hepatocellular carcinoma.

[0034] HBV is a double-stranded DNA virus that replicates via an RNA intermediate and is dependent on viral polymerase. Stable maintenance of HBV in hepatocytes is due to the presence of a covalently closed circular form of viral DNA that is difficult to eradicate.

[0035] Due to its natural tropism and ability to replicate extrachromosomally, HBV as a vector offers important potential advantages for hepatocyte targeting and therapeutic gene delivery. The unique life cycle of HBV provides a unique method for regulating episomal DNA levels through the controlled expression of viral polymerases required for converting RNA intermediates into viral DNA.

[0036] retrovirus Retroviruses are a family of viruses characterized by encoding reverse transcriptases capable of generating DNA copies from RNA templates and by the integration of proviruses into host cell chromosomes. Lentiviruses are a genus of retroviruses that can deliver significant amounts of viral nucleic acid into host cells. Lentiviruses are characterized by their unique ability to infect / transduce non-dividing cells, and after transduction, lentiviruses integrate their nucleic acid into the host cell chromosomes.

[0037] Infectious lentiviruses have three major genes encoding the virulence proteins gag, pol, and env, and two regulatory genes, tat and rev. Depending on the specific serotype and virus, additional accessory genes may be present that encode proteins involved in the regulation, synthesis, and / or processing of viral nucleic acid and other replication functions.

[0038] Furthermore, lentiviruses contain a long terminal repeat (LTR) region, which can be approximately 600 nt in length. The LTR can be divided into U3, R, and U5 regions. The LTR can mediate the integration of retroviral DNA into host chromosomes through the action of integrase. Alternatively, the LTR can be used to circularize viral nucleic acid without integrase functioning.

[0039] Viral proteins involved in the early stages of lentiviral replication include reverse transcriptase and integrase. Reverse transcriptase is an RNA-dependent DNA polymerase encoded by the virus. This enzyme uses the viral RNA genome as a template for synthesis of a complementary DNA copy. Reverse transcriptase also possesses RNase H activity for destruction of the RNA template. Integrase binds both the viral cDNA generated by reverse transcriptase and the host DNA. Integrase processes the LTR before inserting the viral genome into the host DNA. tat acts as a transactivator during transcription, enhancing the initiation and elongation of the RNA copy generated from the viral DNA. The rev-responsive element acts post-transcriptionally to regulate mRNA splicing and transport to the cytoplasm.

[0040] Vector-in-vector technology Disclosed herein is a novel vector-in-vector (VIV) system that combines the desirable features of various viral species to precisely regulate gene delivery and expression. Many viral vectors, including the lentiviral (LV) platform, are known in the art. While LVs offer the advantages of ease of manufacture and flexible targeting, lentiviral transduction, like most other forms of stable transduction, results in chromosomal integration of the LV payload. The chromosomal integration property can be abrogated through mutations that inactivate the viral integrase gene.

[0041] Avoiding chromosomal integration reduces the barrier to in vivo gene delivery. Even integration-defective LVs have a background integration frequency, and any DNA molecules may have a small amount of homology to recombine with host sequences. However, these integration rates are very low and usually not clinically significant.

[0042] For example, papillomavirus ori plus E1 protein, EBV ori plus EBNA-1, or hepadnavirus terminal plus viral polymerase can be added as part of a heterologous viral gene cargo that is not normally maintained episomally. By incorporating this heterologous viral replication machinery into a lentiviral vector, approximately 5 kb of additional cargo space remains, which can accommodate a therapeutic gene of interest.

[0043] Additionally, other control elements can be incorporated into the disclosed virus system. For example, expression of E1, EBNA-1, or HBV polymerase can be driven by an inducible promoter. Many types of inducible promoters are known in the art, and for purposes of the present invention, inducible promoters can include, but are not limited to, promoters that respond to antibiotics (i.e., tetracycline, aminoglycosides, penicillins, cephalosporins, polymyxins, etc.) or other drugs, copper and other metals, alcohol, steroids, light, oxygen, heat, cold, or other physical or chemical stimuli. For example, methods using the disclosed virus system can employ tetracycline-inducible gene expression, which relies on a constant supply of drug for cargo gene expression. The compound used to induce the inducible promoter can be added once or repeatedly, depending on the duration of episome replication and the timing of desired cargo delivery. DNA replication and episome maintenance depend on the induction of E1 and, optionally, E2 or EBNA-1, which in turn depends on the inducer of gene expression (i.e., tetracycline). An exemplary diagram of the VIV system is shown in Figure 1. Another exemplary diagram of the VIV system is shown in Figure 2. As shown in Figure 2, the E1 initiator protein is present and the cargo is GFP under the EF1-HTLV promoter. Another exemplary diagram of the VIV system is shown in Figure 4, in which the gene cargo is represented as a CMV / GFP expression cassette. The cargo gene sequence can be amplified by polymerase chain reaction (PCR) using a synthetic oligonucleotide primer identical to the 5' end of the cargo gene and a synthetic oligonucleotide primer complementary to the 3' end of the cargo gene. The 5' primer can be extended from its 5' end with a recognition site for an endonuclease. The 3' primer can also be extended at its 3' end with a complement for endonuclease recognition. The resulting amplified cargo gene sequence can be annealed to an appropriate vector, such as a lentiviral vector.Non-limiting examples of gene cargo include CMV / VEGF, CMV / anti-epidermal growth factor receptor (EGFR), or miRNA-inhibiting CC chemokine receptor type 5 (CCR5).

[0044] The appropriate expression of cargo can be determined by a suitable assay. For example, the copy number of DNA can be measured by quantitative PCR. The protein product translated from either Vector 1 or Vector 19 (described herein) can be measured, for example, by analytical flow cytometry. ELISA assays can be used to detect the presence of certain cargoes, such as secreted proteins such as VEGF. Western blot techniques can also be used to detect certain cargoes, such as antibodies such as anti-EGFR. In addition, monitoring the reduction of cell surface expression of cargo proteins, such as chemokine receptors such as CCR5, can also be employed.

[0045] As shown in Figure 1, the disclosed VIV can contain at least one gene or sequence of interest. The gene or sequence incorporated into the VIV depends on the purpose of the VIV. Referring to Figure 1, the lentivirus is packaged in an integrase-deficient system, or transduction is performed in the presence of clinical drugs used to block integrase activity (e.g., dolutegravir or raltegravir). If integration fails, the linear, double-stranded vector DNA typically circularizes using the host's enzymatic machinery. A drug-inducible promoter can be activated to express E1 and / or E2 proteins as needed, which then drives DNA replication. The therapeutic cargo is expressed from the cassette. In various embodiments, the compound that induces the inducible promoter (also referred to herein as an "inducer") is turned off or stopped. Turning off the inducer downregulates the synthesis of E1 and / or E2. In further embodiments, production of E1 and / or E2 is effectively stopped. In either event, this reduces the level of episomal DNA and ultimately eliminates the vector construct. Figures 1 and 2 show a VIV system containing E1. Figure 4 shows a VIV system containing both E1 and E2 on a single viral vector. In a preferred embodiment, to express both E1 and E2 from the same mRNA, an internal ribosome entry site (IRES) is added to allow resumption of protein translation.

[0046] For example, the gene encoding platelet-derived growth factor (PDGF) can be incorporated into VIV as a gene, along with the desired shRNA, siRNA, miRNA, and / or other gene silencing RNA, to promote wound healing. The disclosed VIV system is not limited by the type of gene or sequence that can be expressed. Thus, the disclosed VIVs can be used to treat a number of therapeutic or prophylactic genes or sequences, such as antibodies against antigens associated with infectious diseases or cancer (including antigens on replicating pathogens, antigens that are exogenous toxins, and antigens on tumor cells), platelet-derived growth factor, vascular endothelial growth factor, brain-derived growth factor, nerve growth factor, human growth factor, human chorionic gonadotropin, cystic fibrosis transmembrane conductance regulator (CFTR), dystrophin or dystrophin-associated complex, phenylalanine hydroxylase, lipoprotein lipase, α- and / or β-thalassemia, Factor VIII, bone morphogenetic proteins 1-4, cyclooxygenase 2, vascular endothelial growth factor, chemokine receptor CCR5, chemokine receptor CXCR4, chemokine receptor CXCR5, autoimmune diseases involved in colitis, inflammatory bowel disease, or Crohn's disease, or antibodies against antibodies against antigens associated with cancer, including antigens on replicating pathogens, antigens that are exogenous toxins, and antigens on tumor cells, platelet-derived growth factor, vascular endothelial growth factor, brain-derived growth factor, nerve growth factor, human growth factor, human chorionic gonadotropin, cystic fibrosis transmembrane conductance regulator (CFTR), dystrophin or dystrophin-associated complex, phenylalanine hydroxylase, lipoprotein lipase, α- and / or β-thalassemia, Factor VIII, bone morphogenetic proteins 1-4, cyclooxygenase 2, vascular endothelial growth factor, chemokine receptor CCR5, chemokine receptor CXCR4, chemokine receptor CXCR5, autoimmune diseases involved in colitis, inflammatory bowel disease, or Crohn's disease, or antibodies against antibodies against antigens associated with cancer, including antigens against antigens associated with cancer, including antigens on replicating pathogens The vectors may incorporate sequences encoding antisense DNA or RNA against immune antigens, small interfering RNAs involved in addiction including miRNAs that regulate neurasthenia to opiates or alcohol, genes that regulate cell survival including tumor suppressor genes, pro- or anti-apoptotic genes and pro- or anti-autophagy genes, genes encoding radiation resistance factors, genes encoding photoproteins used to track tumor cell metastasis or other cellular trafficking phenomena, or a variety of other therapeutically useful sequences that can be used to condition the body for maximum effectiveness of radiation, surgery, or chemotherapy, or to protect tissue from radiation, surgery, or chemotherapy, to improve organ transplants, or to modify host or graft tissue to suppress hyperresponsiveness, particularly in the airways.

[0047] By maintaining the gene in an episomal form in the VIV system, a safety switch is built in. If the gene product is toxic, DNA replication is halted by withdrawal of the inducer molecule, the episome number subsequently decreases, and the gene and vector are lost. Unlike traditional regulated gene expression, the expression construct disclosed as a safety switch is degraded by endogenous nucleases and diluted with cell division until it is effectively lost, thereby preventing any short- or long-term breakthrough expression.

[0048] Maintaining the gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA in episomal form also allows for widespread and much higher levels of copy number modulation than can be achieved by conventional lentiviral transduction.

[0049] The disclosed VIV system offers many advantages. For example, episomal DNA is less susceptible to chromosomal alterations, which can result in gene silencing compared with conventional transduction vectors. Similarly, VIV episomal DNA vectors support active gene delivery for short- to medium-term periods, at least about 1 to about 4 months, and in some cases longer. In other embodiments of the present invention, episomal DNA vectors support active gene delivery for periods of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks. In other embodiments, episomal DNA vectors support active gene delivery for periods of about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. Any combination of these periods, such as 1 month and 1 week, or 3 months and 2 weeks, can also be used in the methods of the present invention.

[0050] Although there are benefits specifically related to the use of lentiviral carriers for the integration of the disclosed VIV system, the disclosed system is not limited to a single type of viral vector. Any DNA virus or virus that uses a DNA intermediate can be used as a carrier for the integration of VIV replication strategies, including, but not limited to, lentivirus, adeno-associated virus (AAV), adenovirus, vaccinia, herpesvirus, measles virus, hepadnavirus, parvovirus, and murine virus.

[0051] method In some embodiments, the present invention relates to a method of administering a VIV vector to a patient in need thereof, wherein the VIV vector encodes at least one, at least two, at least three, at least four, or at least five genes of interest. Given the versatility and therapeutic potential of the disclosed VIV systems, the VIV systems of the present invention may be used to inhibit a wide variety of antigens, including, but not limited to, antibodies against antigens associated with infectious diseases or toxins produced by infectious pathogens, platelet-derived growth factor, vascular endothelial growth factor, brain-derived growth factor, nerve growth factor, human growth factor, human chorionic gonadotropin, cystic fibrosis transmembrane conductance regulator (CFTR), dystrophin or dystrophin-associated complex, lipoprotein lipase, α- and / or β-thalassemia, Factor VIII, bone morphogenetic proteins 1-4, cyclooxygenase 2, vascular endothelial growth factor, chemokine receptor CCR5, chemokine receptor CXCR4, chemokine receptor CXCR5, antisense DNA or RNA against autoimmune antigens involved in colitis, inflammatory bowel disease, or Crohn's disease. The nucleic acid sequence may encode genes or nucleic acid sequences containing small interfering RNAs involved in addiction, including miRNAs that regulate nervous breakdown to opiates or alcohol, genes that regulate cell survival, including tumor suppressor genes, pro- or anti-apoptotic genes, and pro- or anti-autophagy genes, genes that encode radiation resistance factors, genes that encode photoproteins used to track tumor cell metastasis or other cellular trafficking events, or a variety of other therapeutically useful sequences that can be used to condition the body for maximum effectiveness of radiation, surgery, or chemotherapy, or to protect tissue from radiation, surgery, or chemotherapy, to improve organ transplants, or to modify host or graft tissue to suppress hyper-reactivity, particularly in the airways.

[0052] infectious disease The disclosed compositions and methods can provide protection from disease, particularly for individuals temporarily residing in high-risk areas. Key targets for gene therapy carry significant risks when the gene product is chronically expressed. One exemplary application is the prophylactic delivery of monoclonal antibodies, including protective antibodies against deadly viral agents, necessary to protect individuals traveling within endemic areas (e.g., medics and rescue workers entering Ebola-infected areas). Vaccines have been largely untested for diseases such as Ebola or Lassa fever viruses, dengue fever, chikungunya viruses, or Plasmodium spp., the cause of malaria, and chronic expression of prophylactic antibody genes via the use of integrating vectors carries unknown health risks. Therefore, there is a significant medical need for effective antibody expression, which must be high but transient. The disclosed VIV system and method for delivering high copy numbers of a gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA for a limited period of time fulfill this medical need.

[0053] In one embodiment, the present invention relates to a method of treating, preventing, or minimizing a condition, symptom, or side effect associated with an infectious disease. In some embodiments, the infectious disease can be human immunodeficiency virus (HIV), human T-cell leukemia virus, Ebola virus, Lassa fever virus, dengue fever, Zika virus, malaria, tuberculosis, rabies, vaccinia virus, or other infectious disease. In some embodiments, a VIV vector can be administered prophylactically or after infection with the infectious disease.

[0054] wound healing In another embodiment, the present invention relates to a method for treating, preventing, or minimizing conditions, symptoms, or side effects associated with wound healing. The disclosed compositions can be administered systemically or directly to wounds after accidents, injuries, or surgery. In the case of surgery, VIV vectors can be administered prophylactically to promote healing. In the case of wounds caused by accidents, injuries, or surgery, VIV vectors can be administered some time after wound formation. For example, VIV vectors can be administered within about 1, about 2, about 3, about 4, about 5, about 10, about 12, about 24, about 36, about 48, about 60, about 72, about 84, about 96, about 108, about 120, or about 168 hours after wound formation.

[0055] Another application of the methods and compositions of the present invention is the transient delivery of VIV constructs capable of expressing platelet growth factors to accelerate wound healing. High doses of platelet-derived growth factor (PDGF) are required for a very rapid, but transient, response. The disclosed systems and methods are ideal for this type of application.

[0056] Further short-term applications include the expression of brain-derived growth factor for the intermittent treatment of alcohol abuse, nerve growth factor for spinal cord regeneration, and topical application for skin conditions.

[0057] Bone disease or injury In one embodiment, the disclosed invention relates to a method for enhancing bone healing, comprising identifying a subject with a bone injury and administering to the subject a therapeutically effective amount of a viral delivery system according to the present invention. The viral delivery system comprises a viral carrier, a heterologous viral episomal origin of replication, a sequence encoding an initiator protein specific to the heterologous viral episomal origin of replication, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA, wherein the viral carrier has a defective integrase gene and expression of the sequence encoding the initiator protein specific to the heterologous viral episomal origin of replication is under the control of an inducible promoter. The bone injury may be due to an accident, injury, or surgery, and may be a nonunion or acute fracture or spinal fusion. In some embodiments, the gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA encodes bone morphogenetic protein 1-4, cyclooxygenase-2, or vascular endothelial growth factor.

[0058] In one embodiment, the disclosed invention relates to a method for enhancing bone healing, comprising identifying a subject with a bone disease and administering to the subject a therapeutically effective amount of a viral delivery system according to the present invention. The viral delivery system comprises a viral carrier, a heterologous viral episomal replication origin, a sequence encoding an initiator protein specific to the heterologous viral episomal replication origin, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene silencing RNA, wherein the viral carrier has a defective integrase gene and expression of the sequence encoding the initiator protein specific to the heterologous viral episomal replication origin is under the control of an inducible promoter. The bone disease may be, for example, due to an accident, injury, or surgery, and may be a nonunion or acute fracture or spinal fusion. Furthermore, the bone disease may be due to low bone mineral density, low blood flow to the bone, aging, a hereditary condition, etc. In some embodiments, the gene of interest, shRNA, siRNA, miRNA, and / or other gene silencing RNA encodes bone morphogenetic proteins 1-4, or cyclooxygenase-2, or vascular endothelial growth factor. Inherited genetic disorders [Table 1]

[0059] In another embodiment, the present invention relates to methods for treating, preventing, or minimizing conditions, symptoms, or side effects associated with inherited genetic diseases. Some examples of such inherited genetic diseases are shown in Table 1, using the following nomenclature, along with the causative type of mutation and the chromosome involved: P - point mutation or any insertion / deletion entirely within one gene D - deletion of one or more genes C - Gain, loss, or both of an entire chromosome (see Chromosomal Abnormalities) T-trinucleotide repeat disorders: Genes expand in length

[0060] Current gene therapy involves attempting to edit genomic DNA through gene deletion, replacement, or resequencing.Various gene therapy systems known in the art include Talen, CRISPR-Cas9, zinc finger endonucleases, etc., which rely on the delivery of genetic material by lentiviral transduction.However, unlike the disclosed invention, these systems remain active in cells for extended periods of time because active chromosome modification systems may modify unexpected sites, which can have unexpected consequences, leading to new genetic diseases, including cancer.A truly practical system for modifying host DNA requires transient and well-regulated expression through methods such as those disclosed herein.

[0061] Thus, in one embodiment, the disclosed invention relates to a method for treating a genetic disease, comprising identifying a subject with the genetic disease and administering to the subject a therapeutically effective amount of a viral delivery system according to the present invention. The viral delivery system comprises a viral carrier, a heterologous viral episomal replication origin, a sequence encoding an initiator protein specific to the heterologous viral episomal replication origin, and at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA, wherein the viral carrier has a defective integrase gene, and the expression of the sequence encoding the initiator protein specific to the heterologous viral episomal replication origin is under the control of an inducible promoter. The genetic disease may be, for example, a disease listed in Table 1, and in some embodiments, the gene of interest, shRNA, siRNA, miRNA, and / or other gene-silencing RNA encodes a non-mutated version of a gene listed in Table 1.

[0062] It is also possible to incorporate a guide RNA target sequence into the disclosed VIV vector. Guide RNA is a sequence used to direct gene editing mechanisms to specific sites in the host genome that are mutated or otherwise require modification. By including guide RNA in the cargo of VIV vector, the chromosome section that requires modification can be modified, and the same modification occurs in VIV, accelerating host degradation and / or dilution.Therefore, in certain embodiments of the present invention, the disclosed viral delivery system comprises a viral carrier, a heterologous virus episomal replication origin, a sequence encoding an initiator protein specific to the heterologous virus episomal replication origin, at least one gene of interest, shRNA, siRNA, miRNA, and / or other gene silencing RNA, and at least one guide RNA, wherein the viral carrier has a defective integrase gene, and the expression of the sequence encoding the initiator protein specific to the heterologous virus episomal replication origin is under the control of an inducible promoter.

[0063] Ex vivo modification of cells or tissues In another embodiment, VIV vectors can be used to modify cells or tissues used in disease treatment. Cells can include, but are not limited to, primary cells such as lymphocytes, stem cells, epithelial cells, and neural cells. For example, VIV vectors can be used to modify lymphocytes to redirect specific diseases, including cancer, infectious diseases, or autoimmunity, and when the long-term presence of genetically modified cells poses a health risk. For example, VIV vectors can be used to program pluripotent stem cells that require high levels of transcription factors for a defined interval and when the long-term presence of an integrated viral vector is undesirable. Epithelial cells used for synthetic skin or other applications may require the expression of trophic or growth factors during the initial treatment that would be detrimental to normal tissue function after treatment and are best delivered by VIV vectors.

[0064] Dosage and Formulation The disclosed VIV vectors allow for short-term, medium-term, or long-term expression of the gene or sequence of interest, and for the episomal maintenance of the disclosed vectors. Thus, dosage regimens can vary based on the condition being treated and the method of administration.

[0065] In one embodiment, VIV can be administered to a subject in need thereof at various doses. 6 The subject may be administered an infectious dose (one dose required on average to transduce one target cell). More specifically, the subject may be administered a ≥ 10 7 , ≥ 10 8 , ≥ 10 9 , or ≥ 10 10 It may be administered at an infectious dose. The upper limit of VIV dosing is determined for each disease indication and is based on the toxicity / safety profile of the individual product or product lot.

[0066] Additionally, the VIV of the present invention can be administered once or twice daily. Alternatively, the VIV can be administered to a subject in need thereof once a week, once every two weeks, once every three weeks, once a month, every other month, every three months, every six months, every nine months, once a year, every 18 months, every two years, every 36 months, or every three years.

[0067] In one embodiment, VIV is administered as a pharmaceutical composition. In one embodiment, the pharmaceutical composition containing VIV can be formulated into a wide range of nasal, pulmonary, oral, topical, or parenteral dosage forms for clinical application. Each dosage form can contain various disintegrants, surfactants, fillers, thickeners, binders, wetting agents, or other diluents, or other pharmaceutically acceptable excipients. The pharmaceutical composition containing VIV can also be formulated for injection.

[0068] The VIV compositions may be administered using any pharmaceutically acceptable method, such as intranasal, buccal, sublingual, oral, rectal, ocular, parenteral (intravenous, intradermal, intramuscular, subcutaneous, intracisternal, intraperitoneal), pulmonary, vaginal, topical, localized, post-scarring localized, mucosal administration via aerosol or via buccal or nasal spray formulation.

[0069] Furthermore, the VIV composition can be formulated into any pharmaceutically acceptable dosage form, such as solid dosage forms, tablets, pills, lozenges, capsules, liquid dispersions, gels, aerosols, pulmonary aerosols, nasal aerosols, ointments, creams, semi-solid dosage forms, and suspensions.Furthermore, the composition can be a controlled release formulation, a sustained release formulation, an immediate release formulation, or any combination thereof.Furthermore, the composition can be a transdermal delivery system.

[0070] In another embodiment, a pharmaceutical composition containing VIV can be formulated into a solid dosage form for oral administration, and the solid dosage form can be a powder, granule, capsule, tablet, or pill. In yet another embodiment, the solid dosage form can contain one or more excipients, such as calcium carbonate, starch, sucrose, lactose, microcrystalline cellulose, or gelatin. Furthermore, the solid dosage form can contain a lubricant, such as talc or magnesium stearate, in addition to the excipient. In some embodiments, the oral dosage form can be immediate-release or modified-release. Modified-release dosage forms include controlled- or extended-release, enteric-coated, and the like. Excipients used in modified-release dosage forms are generally known to those skilled in the art.

[0071] In a further embodiment, the pharmaceutical composition comprising VIV can be formulated as a sublingual or buccal dosage form. Such dosage forms include sublingual tablets or solution compositions administered under the tongue, and buccal tablets placed between the cheek and gum.

[0072] In yet a further embodiment, the pharmaceutical composition comprising the VIV can be formulated as a nasal dosage form. Such dosage forms of the present invention include solution, suspension, and gel compositions for nasal delivery.

[0073] In one embodiment, the pharmaceutical composition may be formulated into a liquid dosage form for oral administration, such as a suspension, emulsion, or syrup. In other embodiments, the liquid dosage form may contain various excipients, such as wetting agents, sweeteners, flavoring agents, or preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. In certain embodiments, a composition comprising VIV or a pharmaceutically acceptable salt thereof may be formulated to be suitable for administration to pediatric patients.

[0074] In one embodiment, the pharmaceutical composition may be formulated into a dosage form for parenteral administration, such as a sterile aqueous solution, suspension, emulsion, non-aqueous solution, or suppository. In other embodiments, the non-aqueous solution or suspension may contain propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or an injectable ester such as ethyl oleate. As a base for a suppository, witepsol, macrogol, Tween 61, cocoa oil, lauric oil, or glycerinated gelatin may be used.

[0075] The dosage of the pharmaceutical composition may vary depending on the patient's body weight, age, sex, time and mode of administration, excretion rate, and severity of the disease.

[0076] definition Words not specifically defined herein are understood to have the same meaning as understood by one of ordinary skill in the art.

[0077] As used herein, the term "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If a term is used that is not obvious to a person of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.

[0078] "Treatment" means targeting and combating a condition, i.e., ameliorating or preventing the condition. The particular treatment will thus depend on the condition being targeted and the current or future state of medical therapy and therapeutic approaches. Treatment may be associated with toxicity.

[0079] The terms "administration of" or "administering" an active agent mean providing an active agent of the present invention to a subject in need of treatment in a form that can be introduced into the body of an individual in a therapeutically useful form and in a therapeutically effective amount.

[0080] The term "therapeutically effective amount" refers to a sufficient amount of an active agent of the present invention in a suitable composition and in a suitable dosage form to treat or prevent the symptoms, progression, or onset of complications seen in a patient suffering from a given illness, injury, disease, or condition. The therapeutically effective amount will vary depending on the patient's condition or its severity, and the age, weight, etc., of the subject being treated. The therapeutically effective amount can vary depending on any of a number of factors, including, for example, the route of administration, the condition of the subject, and other factors understood by those skilled in the art.

[0081] The term "treatment" or "treating" generally refers to an intervention in an attempt to alter the natural course of the subject being treated, and may be performed prophylactically or during the course of clinical pathology. Desired effects include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, suppressing, attenuating, or inhibiting any direct or indirect pathological consequences of disease, ameliorating or alleviating the condition, and causing remission or improved prognosis.

[0082] The terms "individual," "host," "subject," and "patient" are used interchangeably herein.

[0083] As used herein, "expression," "expressed," or "encoding" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Expression may include splicing or other forms of post-transcriptional or post-translational modification of the mRNA in eukaryotic cells.

[0084] The following examples are provided to illustrate the present invention. However, the present invention is not limited to the specific conditions or details described in these examples. All published publications referenced herein are specifically incorporated by reference. [Example]

[0085] Example 1 Use of VIV in the treatment of infectious diseases This example demonstrates the use of the disclosed VIV in the treatment of Ebola virus or other infectious diseases, as demonstrated by the non-limiting example illustrated in Figure 1. In this example, at least one of the "cargo" regions shown in Figure 1 encodes an antibody that specifically targets Ebola virus.

[0086] A therapeutically effective amount of VIV encoding an antibody specifically targeting Ebola virus is administered to a subject suspected of having or diagnosed with Ebola, alone or in combination with one or more additional agents for treating or preventing Ebola.The VIV encoding an antibody specifically targeting Ebola virus and / or additional agents are administered orally, intranasally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, topically, systemically, intravenously, subcutaneously, intraperitoneally, or intramuscularly according to a method known in the art or described herein.Subjects are evaluated daily for the presence and / or severity of Ebola-related signs and symptoms, including but not limited to, fever, fatigue, lethargy, weakness, redness of the eyes, joint and muscle pain, headache, nausea, vomiting, bleeding, and death.Treatment is maintained until one or more signs or symptoms of Ebola are improved or eliminated.

[0087] It is reasonably expected that subjects suspected of or diagnosed with Ebola and administered a therapeutically effective amount of a VIV encoding an antibody that specifically targets the Ebola virus will exhibit a reduction in the severity or elimination of one or more symptoms associated with Ebola. Furthermore, it is expected that administering a VIV encoding an antibody that specifically targets the Ebola virus in combination with one or more additional agents will have an additive or synergistic effect.

[0088] These results indicate that VIVs encoding antibodies that specifically target Ebola virus are useful in the treatment of Ebola or other infectious diseases. Thus, VIVs encoding antibodies that specifically target infectious antigens are useful in methods that include administering VIVs to subjects in need thereof for the treatment of infectious diseases.

[0089] Example 2 Use of VIV in preventing infectious diseases This example demonstrates the use of the disclosed VIV in the prevention of Ebola virus or other infectious diseases, as demonstrated by the non-limiting example illustrated in Figure 1. In this example, at least one of the "cargo" regions shown in Figure 1 encodes an antibody that specifically targets Ebola virus.

[0090] A prophylactically effective amount of VIV encoding an antibody specifically targeting the Ebola virus is administered to a subject suspected of being at increased risk of contracting Ebola, alone or in combination with one or more additional agents for treating or preventing Ebola, before entering an area where there is an increased risk of contracting Ebola.The VIV encoding an antibody specifically targeting the Ebola virus and / or the additional agent is administered orally, intranasally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, topically, systemically, intravenously, subcutaneously, intraperitoneally, or intramuscularly according to methods known in the art or described herein.Subjects are evaluated daily for the presence and / or severity of signs and symptoms associated with Ebola, including, but not limited to, fever, fatigue, lethargy, weakness, redness of the eyes, joint and muscle pain, headache, nausea, vomiting, bleeding, and death.Treatment is maintained until one or more signs or symptoms of Ebola are prevented.

[0091] It is reasonably expected that a subject suspected of or diagnosed with Ebola who is administered a prophylactically effective amount of a VIV encoding an antibody that specifically targets the Ebola virus will have a reduced risk of contracting Ebola. Furthermore, it is expected that administering a VIV encoding an antibody that specifically targets the Ebola virus in combination with one or more additional agents will have an additive or synergistic effect in this regard.

[0092] These results indicate that VIVs encoding antibodies that specifically target Ebola virus are useful in preventing Ebola or other infectious diseases. Thus, VIVs encoding antibodies that specifically target infectious antigens are useful in methods that include administering VIVs to subjects in need thereof to prevent infectious diseases.

[0093] Example 3 Use of VIV to enhance wound healing This example demonstrates the use of the disclosed VIV for enhancing wound healing, as demonstrated by the non-limiting example illustrated in Figure 1. In this example, at least one of the "cargo" regions shown in Figure 1 encodes platelet-derived growth factor (PDGF).

[0094] A subject having a wound (e.g., due to an accident, injury, or surgery) is administered a therapeutically effective amount of VIV encoding platelet-derived growth factor (PDGF), alone or in combination with one or more additional agents for treating or sterilizing the wound. PDGF and / or other active substance are administered orally, intranasally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, locally, systemically, intravenously, subcutaneously, intraperitoneally or intramuscularly according to the method known in the art or as described herein.Evaluate the subject every day to determine the state of wound.Treatment is maintained until the time when wound heals and scarring is minimized.

[0095] It is reasonably expected that a subject having a wound and receiving a therapeutically effective amount of VIV PDGF will exhibit enhanced wound healing. Furthermore, it is expected that administering VIV encoding PDGF in combination with one or more additional agents will have additive or synergistic effects.

[0096] These results show that VIV encoding PDGF is useful for enhancing wound healing.Therefore, VIV encoding PDGF or similar genes are useful in the method comprising administering VIV to a subject in need thereof for treating wounds.

[0097] Example 4 Use of VIV in the treatment of bone injuries This example demonstrates the use of the disclosed VIV in the treatment of bone injury, as demonstrated by the non-limiting example illustrated in Figure 1. In this example, at least one of the "cargo" regions shown in Figure 1 encodes vascular endothelial growth factor (VEGF).

[0098] The subject suspected of having bone injury or diagnosed as having bone injury is administered a therapeutically effective amount of VIV encoding vascular endothelial growth factor (VEGF), alone or in combination with one or more additional active substances for treating bone injury.The VIV encoding VEGF and / or additional active substances are administered orally, intranasally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, locally, systemically, intravenously, subcutaneously, intraperitoneally or intramuscularly according to the method known in the art or described herein.The subject is evaluated weekly for the presence and / or severity of the signs and symptoms associated with bone injury to determine the speed and strength of healing.Treatment is maintained until the time when bone is healed.

[0099] It is reasonably expected that subjects suspected of having or diagnosed with bone injury and administered a therapeutically effective amount of VIV encoding VEGF will show a reduction in the severity of the injury and an enhancement in healing.Furthermore, it is expected that administering VIV encoding VEGF in combination with one or more additional agents will have an additive or synergistic effect.

[0100] These results indicate that VIV encoding VEGF is useful in the treatment of bone injury or disease.Therefore, VIV encoding VEGF or similar gene is useful in the method comprising administering VIV to a subject in need thereof for the treatment of bone injury or disease.

[0101] Example 5 Use of VIV in the treatment of genetic disorders This example demonstrates the use of the disclosed VIV in the treatment of cystic fibrosis (CF) or other inherited genetic diseases, as demonstrated by the non-limiting examples illustrated in Figure 1. In this example, at least one of the "cargo" regions shown in Figure 1 encodes the cystic fibrosis transmembrane conductance regulator (CFTR).

[0102] The subject suspected of having (CF) or diagnosed with (CF) is administered a therapeutically effective amount of VIV encoding cystic fibrosis transmembrane conductance regulator (CFTR), alone or in combination with one or more additional agents for treating CF.The VIV encoding CFTR and / or additional agents are administered orally, intranasally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, locally, systemically, intravenously, subcutaneously, intraperitoneally, or intramuscularly according to the method known in the art or described herein.Subject is evaluated weekly for the presence and / or severity of the signs and symptoms associated with CF, including but not limited to, poor growth, persistent cough, thick sputum and mucus, wheezing, shortness of breath, reduced exercise capacity, repeated lung infections, nasal inflammation, oily stool, intestinal obstruction, and poor weight gain.Treatment is maintained until one or more signs or symptoms of CF are improved or eliminated.

[0103] It is reasonably expected that subjects suspected of or diagnosed with CF and administered a therapeutically effective amount of a VIV encoding CFTR will exhibit a reduction in the severity or elimination of one or more symptoms associated with CF. Furthermore, it is expected that administering a VIV encoding CFTR in combination with one or more additional agents will have additive or synergistic effects.

[0104] These results indicate that VIV encoding CFTR is useful in the treatment of CF or other inherited genetic diseases. Thus, VIV encoding a mutated or otherwise defective gene associated with a genetic disease is useful in methods comprising administering VIV to a subject in need thereof for the treatment of the genetic disease.

[0105] Example 6 Use of E1-containing VIV to express cargo A vector containing the green fluorescent protein gene (GFP) as cargo was constructed according to Figure 2. DNA containing the complete locus control region and E1 protein of human papillomavirus type 16 (NCBI accession number U89348) was chemically synthesized. Individual segments and / or coding sequences were first synthesized. These were amplified by polymerase chain reaction (PCR) using a synthetic oligonucleotide primer identical to the 5' end of the green fluorescent protein gene and a synthetic oligonucleotide primer complementary to the 3' end of the green fluorescent protein gene. The 5' primer extended from its 5' end with a recognition site for BamHI or EcoRI endonuclease. The 3' primer extended at its 3' end with the complement of the BamHI or EcoRI endonuclease recognition site. The resulting amplified green fluorescent protein gene sequence was then digested with BamHI and EcoRI restriction endonucleases.

[0106] The lentiviral vector was obtained from System Biosciences, Inc. The plasmid was cleaved with BamHI and EcoRI enzymes and mixed with the over-amplified green fluorescent protein gene sequence at a ratio of 1:3 insert to vector.

[0107] Enzyme activity was then stopped by heat inactivation for 20 minutes at 70° C. The mixture was cooled to room temperature and allowed to anneal.

[0108] The annealing reaction was carried out using bacteriophage T4 DNA ligase for 30 minutes at room temperature. 2.5 microliters of the resulting ligation mixture was added to 25 microliters of STBL3 competent bacterial cells.

[0109] Transfection was then carried out by a brief (1 min) heat shock at 42 degrees Celsius.

[0110] Bacterial cells were streaked onto agar plates containing ampicillin to obtain bacterial cultures, which were grown in Luria broth.

[0111] To confirm the insertion of the amplified green fluorescent protein gene sequence into the lentiviral vector packaging plasmid, DNA was extracted from the bacterial culture and purified by standard methods. The purified DNA was digested with the same endonucleases used to generate the construct. Fragment lengths were analyzed by agarose gel electrophoresis, and the amplified green fluorescent protein gene sequence was verified by DNA sequencing using specific primers obtained from Eurofins MWG Operon LLC.

[0112] Lentiviral vector stocks were generated as follows: At least two lentiviral packaging plasmids plus a cargo plasmid were cotransfected into HEK cells expressing viral genes and genomic RNA, assembled into integrase-deficient lentiviral particles, and released into the culture medium. Cell-free supernatants were generated and harvested at intervals of 3–10 days posttransfection. Lentiviral particles were purified by standard procedures, including a combination of methods that may include centrifugation, transient flow filtration, size-exclusion chromatography, size-exclusion filtration, or ion-exchange chromatography. The concentration and biological activity (transducing units per ml) of each stock were determined.

[0113] Mammalian cells, including 293T cells, were used to test the formation, copy number, and expression of lentivirus-derived episomes. 293T cells were transduced with integrase-deficient lentiviral particles in the presence of polybrene at multiplicities of infection ranging from 1 to 10. Unabsorbed virus was removed by washing the cells 3 hours after application, and the cells were cultured for 3 days. Cells were observed under a fluorescent microscope, and GFP-expressing cells were counted. Untransduced 293T cells were used as a negative control. Data were reported as GFP-positive cells per 100 viable cells in culture. A minimum of 300 cells were counted per microscopic field, with 5–10 fields counted for each replicate experiment. Four independent transduction experiments, including one negative control and three replicate experiments, were performed to determine the frequency of transduced cells. The data are shown in Figure 3, showing GFP expression across three replicate experiments.

[0114] Example 7 Use of VIV containing E1 and E2 to express cargo Vector 19 is constructed to contain both the E1 and E2 initiator proteins, as shown in Figure 4. In this example, the gene cargo is represented by a CMV / GFP expression cassette, and an appropriate inducible promoter is also selected to allow for the exogenous addition of compounds capable of inducing the inducible promoter.

[0115] 293T cells are transduced with vector 19 at a multiplicity of infection ranging from 1 to 20 transducing units per cell. After 3 hours, cells are washed with medium to remove unadsorbed virions and returned to culture. 12 to 24 hours after transduction, cells are treated with at least one dose of a compound capable of inducing an inducible promoter. Upon addition of a compound capable of inducing an inducible promoter, E1 and E2 mRNAs are transcribed from the episome, assembled into the locus control region fragment 2 (LCR / F2), and assembled to trigger DNA replication. The lentivirus-derived episome begins to disintegrate approximately 24 to 36 hours after cessation of promoter induction. Protein products from the cargo within vector 19 are measured by analytical flow cytometry.

[0116] While certain preferred embodiments of the present invention have been described and specifically exemplified herein, it is not intended that the invention be limited to such embodiments, and various modifications thereto may be made without departing from the scope and spirit of the invention. In certain embodiments, for example, the following items are provided: (Item 1) (a) a viral carrier having a defective integrase gene; (b) heterologous viral episomal origin of replication; (c) a sequence encoding at least one initiator protein specific for a heterologous viral episomal origin of replication, wherein expression of the sequence encoding at least one initiator protein specific for the heterologous viral episomal origin of replication is under the control of an inducible promoter; and (d) at least one gene of interest, shRNA, siRNA, miRNA, or other gene-silencing RNA; A viral delivery system comprising: (Item 2) Item 1, wherein the viral carrier is a lentivirus. (Item 3) 2. The viral delivery system of item 1, wherein the heterologous viral episomal origin of replication is derived from a papillomavirus. (Item 4) 4. The viral delivery system of item 3, wherein the heterologous viral episomal origin of replication is derived from a bovine papillomavirus. (Item 5) 4. The viral delivery system of item 3, wherein the heterologous viral episomal origin of replication is derived from a human papillomavirus. (Item 6) 2. The viral delivery system of item 1, wherein the at least one initiator protein specific for the heterologous viral episomal replication origin is E1. (Item 7) 2. The viral delivery system of item 1, wherein the at least one initiator protein specific for the heterologous viral episomal replication origin is E2. (Item 8) 2. The viral delivery system of item 1, comprising two initiator proteins specific for the heterologous viral episomal replication origin. (Item 9) 9. The viral delivery system of item 8, wherein the two initiator proteins specific for the heterologous viral episomal replication origin are E1 and E2. (Item 10) Item 2. The viral delivery system according to item 1, wherein the heterologous viral episomal origin of replication is derived from Epstein-Barr virus. (Item 11) 11. The viral delivery system of item 10, wherein the initiator protein specific for the heterologous viral episomal replication origin is EBNA-1. (Item 12) 2. A pharmaceutical composition comprising the viral delivery system of item 1 and at least one pharmaceutically acceptable carrier. (Item 13) (a) identifying a subject in need of treatment or prevention of a disease; and (b) administering a therapeutically effective amount of the viral delivery system to the subject. 10. A method for treating or preventing a disease, comprising: (i) a viral carrier having a defective integrase gene; (ii) a heterologous viral episomal origin of replication; (iii) a sequence encoding at least one initiator protein specific for a heterologous viral episomal origin of replication, wherein expression of the sequence encoding the at least one initiator protein is under the control of an inducible promoter; and (iv) at least one gene of interest, shRNA, siRNA, miRNA, or other gene-silencing RNA; A method comprising: (Item 14) 14. The method of claim 13, wherein the disease is an infectious disease. (Item 15) 15. The method of claim 14, wherein the infectious disease is Ebola virus or Lassa virus. (Item 16) 16. The method of claim 15, wherein the gene of interest, shRNA, siRNA, miRNA, or other gene silencing RNA encodes an antibody specific to Ebola virus or Lassa virus. (Item 17) 14. The method of item 13, wherein the disease is a genetic disease or disorder. (Item 18) 14. The method of claim 13, wherein the genetic disease is alcohol abuse. (Item 19) 14. The method of claim 13, wherein the gene of interest, shRNA, siRNA, miRNA, or other gene silencing RNA encodes a brain-derived growth factor. (Item 20) (a) identifying a subject having a neurological injury; and (b) administering a therapeutically effective amount of the viral delivery system to the subject. 1. A method of treating nerve injury, comprising: (i) a viral carrier having a defective integrase gene; (ii) a heterologous viral episomal origin of replication; (iii) a sequence encoding at least one initiator protein specific for a heterologous viral episomal origin of replication, wherein expression of the sequence encoding the at least one initiator protein is under the control of an inducible promoter; and (iv) at least one gene of interest, shRNA, siRNA, miRNA, or other gene-silencing RNA; A method comprising: (Item 21) 21. The method of claim 20, wherein the gene of interest, shRNA, siRNA, miRNA, or other gene silencing RNA is a nerve growth factor. (Item 22) (a) identifying a subject having a wound; and (b) administering a therapeutically effective amount of the viral delivery system to the subject. 1. A method of enhancing wound healing, comprising administering to a subject a viral delivery system comprising: (i) a viral carrier having a defective integrase gene; (ii) a heterologous viral episomal origin of replication; (iii) a sequence encoding at least one initiator protein specific for a heterologous viral episomal origin of replication, wherein expression of the sequence encoding the at least one initiator protein is under the control of an inducible promoter; and (iv) at least one gene of interest, shRNA, siRNA, miRNA, or other gene-silencing RNA; A method comprising: (Item 23) 23. The method of claim 22, wherein the gene of interest, shRNA, siRNA, miRNA, or other gene silencing RNA is platelet-derived growth factor or vascular endothelial growth factor. (Item 24) 23. The method of claim 22, wherein the wound is due to an accident or injury. (Item 25) 23. The method of claim 22, wherein the wound is surgical. (Item 26) 23. The method of claim 22, wherein the wound is a burn. (Item 27) 23. The method of claim 22, wherein the wound is a bony nonunion.

Claims

[Claim 1] A composition as described in the specification.

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