Vectors in gene therapy

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The state of the art of adeno associated virus based vectors in gene therapy

The state of the art of adeno associated virus based vectors in gene therapy

This increasing interest on AAV is justified by its character- istic features that distinguish it from many other viral vec- tor systems, such as retro/lentiviral and adenoviral vectors, and turn it into a very attractive tool for gene ther- apy. These features, as mentioned above, include: (1) its nonpathogenicity and nonimmunogenicity as well as its heat stability and resistance to solvents and to changes in pH and temperature [15]; (2) AAV vectors only retain about 300 nucleotides of viral sequence in the form of nontranscribed ITRs, which greatly improves its safety for human clinical applications by reducing the risk of recom- bination with wild-type virus. Moreover, lack of viral cod- ing sequences extends the duration of gene expression as no viral gene products are expressed in target cells, which reduces the risk of eliciting a cellular immune response; (3) AAV vectors have a broad host and cell type tropism range and transduce both dividing and nondividing cells in vitro and in vivo. Furthermore, the recent discovery of novel AAV serotypes will expand even more the universe of potential target organs, tissues and cells; and (4) AAV vectors maintain (over several years) high levels of gene expression in vivo, in the absence of a significant immune response to the transgene product. This is a major require- ment for gene therapy approaches for some diseases, and constitutes the most promising and distinguishing fea- tures of AAV vectors.
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Retroviral vectors and transposons for stable gene therapy: advances, current challenges and perspectives

Retroviral vectors and transposons for stable gene therapy: advances, current challenges and perspectives

However, it is important to note that MLV vectors have the biological disadvantage that they are unable to effi- ciently transduce non-dividing or slowly dividing cells. As a result, MLV vectors were gradually replaced by len- tiviral vectors based on HIV-1, which can integrate in the host genome of non-dividing cells nearly as well as in dividing cells [59]. Lentiviral vectors finally entered clini- cal trials a few years ago, and several gene therapy pro- tocols are currently underway with impressive results around the world. In 2001, the first human subject was treated with lentiviral vectors. Autologous CD4+ cells from HIV  +  patients were transduced with a lentiviral vector based on HIV-1, containing antisense sequences against the HIV-1 envelope gene [60]. Thereafter, gene therapy with lentivectors was extended to the pre-clinical study of several monogenic diseases, such as hemophilia [61], a X-linked bleeding disorder caused by mutations in Factor VIII or Factor IX genes and; metachromatic leu- kodystrophy [62], caused by arylsulfatase A deficiency. In 2007 the firsts clinical trials of monogenic diseases, adrenoleukodystrophy and beta-thalasemia, took place [63–65]. More recently, gene transfer via lentiviral vec- tors was shown to revert the disease state in the long term in patients of metachromatic leukodystrophy and Wiskott-Aldrich syndrome [66, 67]. These studies indi- cate that lentiviral-based gene therapy is a safe and effec- tive approach to treat distinct diseases [65]. Furthermore, concerning ADA-SCID therapy, it is important to high- light that the trials using retroviral vectors presented similar survival rates to those achieved in hematopoietic stem cell (HSC) transplantation in patients undergoing an HLA-matched donor transplant [68]. Until now, there is only one clinical study reporting negative side effects due to insertional mutagenesis of lentivirus based vectors (as detailed in 4.3.1) and, although some clonal patterns in hematopoietic reconstitutions were suggested, this clonal skewing with transduced cells led to no evident clinical implications [69, 70].
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Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors

Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors

Although long-lasting factor concentrates have im- proved efficacy of protein replacement therapy, and FVIII-mimetic bispecific antibody is promising as an alternative treatment especially for patients with in- hibitory antibodies, these therapies require frequent, at least weekly dosing of costly reagents. With the re- cent successes in clinical trials of AAV-mediated gene transfer, it is anticipated that gene therapy will be the next generation medicine for hemophilia treatment. The potential obstacles of AAV-mediated gene ther- apy include unpredictable events of transaminase ele- vation, potential requirement of repeated treatment that cannot be performed due to pre-existing anti- AAV immune responses, and the prohibitively high cost of high-titer vectors needed for hemA treatment. The recently developed novel strategy of IO delivery of F8-LVs [10] can efficiently transduce bone marrow cells and express FVIII in HSCs without the requirement of using potentially toxic, myelosuppressive pre-conditioning methods and the risk of inducing thrombocytopenia. Al- though ubiquitous expression of FVIII and secretion into the circulation induced high-titer inhibitory antibody pro- duction and eliminated functional FVIII activity, a single IO infusion of G-F8-LVs driven by a megakaryocyte- specific promoter produced long-term stable expression of hFVIII in platelets and corrected hemophilia phenotype for long term. Most significantly, this strategy is proven successful in HemA mice with pre-existing inhibitory anti- bodies. Use of combination of pharmacological agents
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Long-term gene therapy with Del1 fragment using nonviral vectors in mice with explanted tumors

Long-term gene therapy with Del1 fragment using nonviral vectors in mice with explanted tumors

Abstract: Cancer gene therapy using nonviral vectors is useful for long periods of treatment because such vectors are both safe and inexpensive, and thus can be used repeatedly. It has been reported that gene therapy with an E3C1 fragment of Del1 in a mouse explanted tumor model improved prognosis. The present study aimed to analyze the long-term effects of repeated non- viral gene transfer of E3C1. Mice with explanted tumors of SCCKN cells, a human squamous carcinoma, were treated with a plasmid encoding E3C1. Plasmids were injected locally every week using a transfection reagent. Control mice treated with mock DNA started to be euthanized on day 18, because the tumors had grown to over 15% of the body weight, and all of them had died by day 43. On the other hand, the tumors in two of ten mice treated with E3C1 had disappeared. The other eight mice started to be euthanized on day 46 and eight of ten mice had been euthanized by day 197. After 18 days of therapy, the tumor volume of control mice was 2,804±829 mm 3 and that of the E3C1 mice was 197±159 mm 3 . Histochemical studies showed
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E- vectors: development of novel self-inactivating and self-activating retroviral vectors for safer gene therapy.

E- vectors: development of novel self-inactivating and self-activating retroviral vectors for safer gene therapy.

We have developed novel self-inactivating and self-activating retroviral vectors based on the previously observed high-frequency deletion of direct repeats. We constructed spleen necrosis virus (SNV)-based viral vectors that contained large direct repeats flanking the viral encapsidation sequence (E). A large proportion of the proviruses in the target cells had E and one copy of the direct repeat deleted. Direct repeats of 1,333 and 788 bp were deleted at frequencies of 93 and 85%, respectively. To achieve a 100% deletion efficiency in target cells after ex vivo infection and drug selection, we constructed a self-activating vector that simultaneously deleted E and reconstituted the neomycin phosphotransferase gene. Selection of the target cells for resistance to G418 (a neomycin analog) ensured that all integrated proviruses had E deleted. The proviruses with E deleted were mobilized by a replication-competent virus 267,000-fold less efficiently than proviruses with E. We named these self-inactivating vectors E 2 (E-minus) vectors. These vectors should increase the safety of retroviral vector-mediated gene therapy by preventing the spread of vector sequences to nontarget cells in the event of coinfection with helper virus. We propose that direct-repeat deletions occur during RNA-dependent DNA synthesis and suggest that template switches occur without a requirement for RNA breaks. The minimum template dissociation frequency was estimated as 8%/100 bp per replication cycle. These vectors demonstrate that large direct repeats and template-switching properties of reverse transcriptase can be utilized to delete any sequence or reconstitute genes during retroviral replication.
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Gene Therapy Vectors Based on Adeno-Associated Virus Type 1

Gene Therapy Vectors Based on Adeno-Associated Virus Type 1

The complete sequence of adeno-associated virus type 1 (AAV-1) was defined. Its genome of 4,718 nucleotides demonstrates high homology with those of other AAV serotypes, including AAV-6, which appears to have arisen from homologous recombination between AAV-1 and AAV-2. Analysis of sera from nonhuman and human primates for neutralizing antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i) NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates, while the reverse is true in humans; and (ii) sera from 36% of nonhuman primates neutralized AAV-1 but not AAV-2, while sera from 8% of humans neutralized AAV-2 but not AAV-1. An infectious clone of AAV-1 was isolated from a replicated monomer form, and vectors were created with AAV-2 inverted terminal repeats and AAV-1 Rep and Cap functions. Both AAV-1- and AAV- 2-based vectors transduced murine liver and muscle in vivo; AAV-1 was more efficient for muscle, while AAV-2 transduced liver more efficiently. Strong NAB responses were detected for each vector administered to murine skeletal muscle; these responses prevented readministration of the same serotype but did not substantially cross-neutralize the other serotype. Similar results were observed in the context of liver-directed gene transfer, except for a significant, but incomplete, neutralization of AAV-1 from a previous treatment with AAV-2. Vectors based on AAV-1 may be preferred in some applications of human gene therapy.
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Multicomponent nanoparticles as nonviral vectors for the treatment of Fabry disease by gene therapy

Multicomponent nanoparticles as nonviral vectors for the treatment of Fabry disease by gene therapy

In order to better know the behavior of the vectors in Hep G2 cells, we performed transfection studies with nano- particles bearing the pCMS-EGFP plasmid that encodes the green fluorescent protein. In the process of developing new vectors for gene therapy, reporter genes such as EGFP, ß-galactosidase, or luciferase plasmids are frequently used since its evaluation, both in vitro and in vivo, is simple and quick. In our case, EGFP expression could be easily measured by flow cytometry and/or fluorimetry in order to determine both the percentage of transfected cells and the amount of protein produced. Transfection of the Hep G2 cells with the vectors bearing the pCMS-EGFP plasmid (Figure 3) revealed that all formulations assayed were able to produce transfection. With the protamine-DNA-SLN and dextran-protamine-DNA-SLN formulations, the percentage of transfected cells was twice that of the DNA-SLN vector (P , 0.05).
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Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy.

Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy.

Recombinant adenoviruses with E1 sequences deleted efficiently transfer genes into a wide variety of target cells. Antigen- and nonantigen-specific responses to the therapy lead to toxicity, loss of transgene expression, and difficulties with vector readministration. We have created new cell lines that allowed the isolation of more disabled adenovirus vectors that have both E1 and E4 deletions. Studies with murine models of liver-directed gene therapy indicated that the E1- and E4-deleted vector expresses fewer virus proteins and induces less apoptosis, leading to blunted host responses and an improved safety profile. The impact of the E4 deletion on the stability of vector expression was confounded by immune responses to the transgene product, which in this study was b -galactosidase. When transgene responses were eliminated, the doubly deleted vector was substan- tially more stable in mouse liver than was the E1-deleted construct. These studies indicate that adenovirus vectors with both E1 and E4 deletions may have advantages in terms of safety and efficacy over first-generation constructs for liver-directed gene therapy.
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The use of high-frequency ultrasound imaging and biofluorescence for in vivoevaluation of gene therapy vectors

The use of high-frequency ultrasound imaging and biofluorescence for in vivoevaluation of gene therapy vectors

F-FDG-microPET. They also showed that 18 F-FDG- microPET was not so useful for determining tumour size, although there was some correlation (R 2 = 0.75). This was similar to our findings with biofluorescence imaging. As with our study, this functional tumour imaging modality is useful for metabolic imaging and should give an indication of the effect of a gene therapy vector on tumour viability. In the current study, HF-US accurately showed the slower tumour growth of the vector- transfected cell line compared to the parental cell line, as predicted from in vitro cell growth curves [16]. However, lobe formation was unexpected. We are currently investi- gating whether this is due to the GFP gene or other com- ponents of the vector backbone. We also demonstrated the utility of the different greyscale textures in monitoring different patterns of growth. The discrimination of areas of necrosis and high vascularity (using contrast agents) was also possible. This should allow real-time monitoring of agents that currently have little apparent effect on tumour volume but may have useful effects of anti- angiogenesis or inducing cell senescence. HF-US would be of particular use for very small xenografts, orthotopic models to in transgenic mice such as the Apc Min/+ mouse, where callipers cannot access the tumour. Indeed, gene therapy vectors are also used in non-cancer applications such as diabetes or organ regeneration, where callipers may not be used to measure disease progress or regres- sion. In these cases, HF-US would be invaluable in moni- toring progress longitudinally without sacrifice of mice.
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Progresses towards safe and efficient gene therapy vectors

Progresses towards safe and efficient gene therapy vectors

and cocal vesiculovirus glycoprotein [142]. Cells of the immune system, such as lymphocytes, have been targeted with vectors pseudotyped with glycoproteins derived from Measles virus [143] or Tupaia paramyxovirus [144]. Such capsid chimeric vectors might have a relevant clinical importance for lymphocyte-gene therapy and immunotherapy. Other efforts have been directed to re-target retroviral vectors towards airway epithelial cells, which might have an impact in treatment options for lung diseases such as cystic fibrosis. The property of Sendai virus (SeV) to bind sialic acid and cholesterol receptors has been exploited to efficiently transduce epithelial cells by pseudotyping the simian immunodeficiency virus (SIV) capsid with the HN and F envelope proteins of SeV [145, 146]. The attachment and fusion proteins of Nipah virus can be used to specifically transduce endothelial cells [147]. In addition, the xenotropic and polytropic retrovirus receptor 1 (XPR1) expressing cells, such as pancreas, kidney, heart and hematopoietic cells can be successfully targeted with retroviral vectors pseudotyped with the murine leukemia virus-related virus (XMRV) Env protein [148]. Therefore, when one wants to design a LV vector that is intended to transduce a specific cell type, searching for other viral glycoproteins that have a receptor on that particular cell type, is the first step in the pseudotyping method of altering the vectors’ natural tropism. Likewise, ligands with receptor specificity could also be fused to pseudotyped retroviral vectors to improve transduction of target cells in a cell-type specific manner [149].
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Alpharetroviral Vectors: From a Cancer-Causing Agent to a Useful Tool for Human Gene Therapy

Alpharetroviral Vectors: From a Cancer-Causing Agent to a Useful Tool for Human Gene Therapy

Promoter insertion, promoter activation and gene transcript truncation are the three most prevalent mechanisms of retroviral vector genotoxicity described to date. While these mechanisms have been described individually for the sake of clarity, insertional mutagenic events show a higher layer of complexity. For example, a single integration may affect more than one gene and induce transcriptional deregulation by more than one mechanism simultaneously [30]. Importantly, our current understanding of genotoxicity mechanisms allows several prevention strategies to be envisioned. Since insertional deregulation of cellular transcription is dependent on the presence of strong promoter/enhancer sequences and splice sites, optimized vector design should omit these elements. In addition, integration target sites influence genotoxicity, with potentially detrimental integrations occurring near or within genes, especially proto-oncogenes and tumor suppressor genes. In this regard, genome-wide studies of retroviral DNA integration have elucidated distinct integration target site preferences for different retroviral vectors. While gammaretroviral vectors preferentially integrate in the proximity of transcription start sites, CpG islands, and genes with implications in cancer, lentiviral vectors tend to integrate within transcription units of actively transcribed genes [27,31–41]. These integration target site preferences are influenced by retrovirus-specific interactions of the retroviral integrase with cellular tethering factors, such as lens-epithelium-derived growth factor/p75 (LEDGF) in the lentiviral [42–46] and bromodomain and extraterminal domain (BET) proteins in the gammaretroviral context [47–49]. These tethering factors direct retroviral integrations to specific regions in the genome and thus largely contribute to integration target site preferences. There have been attempts to modify retroviral vectors and/or their tethering factors with the aim to obtain potentially safer integration characteristics [50–56]. However, some of these modifications suffered from reduced gene transfer efficacy or incomplete redirection of integration site preference and clinical applicability remains to be shown. Importantly, in contrast to gammaretroviral and lentiviral vectors, alpharetroviral vectors have a relatively neutral integration spectrum [57–61]. It is currently unknown whether alpharetroviral integration follows a pattern yet to be identified or if it occurs independently from tethering factors. Nevertheless, the comparatively neutral integration pattern of alpharetroviral vectors renders them less genotoxic [59–61] and thus increases their therapeutic value for future human gene therapy.
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The use of high-frequency ultrasound imaging and biofluorescence for in vivo evaluation of gene therapy vectors

The use of high-frequency ultrasound imaging and biofluorescence for in vivo evaluation of gene therapy vectors

F-FDG-microPET. They also showed that 18 F-FDG- microPET was not so useful for determining tumour size, although there was some correlation (R 2 = 0.75). This was similar to our findings with biofluorescence imaging. As with our study, this functional tumour imaging modality is useful for metabolic imaging and should give an indication of the effect of a gene therapy vector on tumour viability. In the current study, HF-US accurately showed the slower tumour growth of the vector- transfected cell line compared to the parental cell line, as predicted from in vitro cell growth curves [16]. However, lobe formation was unexpected. We are currently investi- gating whether this is due to the GFP gene or other com- ponents of the vector backbone. We also demonstrated the utility of the different greyscale textures in monitoring different patterns of growth. The discrimination of areas of necrosis and high vascularity (using contrast agents) was also possible. This should allow real-time monitoring of agents that currently have little apparent effect on tumour volume but may have useful effects of anti- angiogenesis or inducing cell senescence. HF-US would be of particular use for very small xenografts, orthotopic models to in transgenic mice such as the Apc Min/+ mouse, where callipers cannot access the tumour. Indeed, gene therapy vectors are also used in non-cancer applications such as diabetes or organ regeneration, where callipers may not be used to measure disease progress or regres- sion. In these cases, HF-US would be invaluable in moni- toring progress longitudinally without sacrifice of mice.
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HIV-Derived Lentiviral Vectors: Current Progress toward Gene Therapy and DNA Vaccination

HIV-Derived Lentiviral Vectors: Current Progress toward Gene Therapy and DNA Vaccination

Lentiviral vectors are promising gene delivery tools capable of transducing a variety of dividing and non-dividing cells, including pluripotent stem cells which are refractory for transduction by murine retroviruses. Although there is a growing debate on the safety of lentiviral vectors for gene transfer, in particular for those derived from human immunodeficiency viruses, type one (HIV-1) and type two (HIV-2), these vectors are envisioned to possess several advantages. Importantly, they can be utilized not only for transducing specific target cells or for in vivo gene therapy of HIV infection and acquired immunodeficiency syndrome (AIDS), but also in a pseudotype recombinant form can be used for different target cells including neurological and cancer cells. For HIV-2, the most com- pelling advantages are: (i) its reduced ability to recombine with resident HIV-1 genome; (ii) its ability to induce in recipients antibodies which can be distinguished from host immune response to HIV-1; (iii) HIV-2 is apparently less pathogenic; and (iv) may downregulate HIV-1 expression. This review will summarize new developments on HIV-1 vectors, while focusing on alternate strategies toward developing HIV-2-based vectors. Iran. Biomed. J. 2: 95-103, 1998
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Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors

Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors

An estimated 30.3 million US Americans are affected by either type 1 or type 2 diabetes mellitus (1). Vari- ous strategies to cure diabetes have been evaluated over the years with limited success. For example, trans- plantation of cadaveric human islets into the hepatic duct has been used to replace β cells in type 1 diabetic patients, so far with low efficiencies (reviewed in ref. 2). Islets are highly vascularized and require large amounts of oxygen to survive. Because revascularization of the transplanted islets takes several weeks, those transplants suffer from a large number of cell death due to oxygen deprivation. In order to improve graft survival and function, different approaches of ex vivo gene therapy, either by supplying or repressing certain transcription factors using different viral or nonviral delivery systems (for example adeno-associated virus (AAV), adenovirus, lentivirus, various lipids, or nonlipid polymers), may be used (reviewed in ref. 3). Recombinant AAV–mediated (rAAV-mediated) overexpression of Follistatin, a protein with important roles in the proper functioning of the reproductive, endocrine, and muscoskeletal systems, has been shown to promote β cell proliferation and maintain pancreatic islet mass in a diabetic mouse model (4). In addi- tion to the approaches described above, it is of the utmost importance to prevent loss of the transplanted islets due to recurrent autoimmune destruction. Recently, a study described the use of rAAV to overexpress Igf1 in diabetic mice (5). Igf1 is a prosurvival factor and β cell mitogen that has important roles in β cell maturation and function and is also involved in the interplay between the endocrine and the immune sys- tem. Over a period of 30 weeks, this gene therapy strategy was successfull in counteracting progression to autoimmune diabetes. One strategy for diabetes treatment is the conversion of α cells or other endocrine or exocrine pancreatic cell types into β cells (reviewed in ref. 6). Cell conversion has been achieved by overexpression or repression of certain transcription factors, such as Pdx1, Ngn3, MafA, Pax4, and Arx (7– 15). Most of those in vivo studies involved transgenic mouse models or used adenoviral vectors to deliver expression cassettes. However, rAAV vectors packaged with AAV8 capsid were also employed (11), as this serotype had previously been found to transduce murine β cells with high efficiency (16, 17). Recently, i.p. delivery of an AAV8-based vector expressing IL-2 under control of the β cell type–specific insulin promoter has been described to achieve highly specific transduction of β cells in mice (18). Another study found that
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Incidence and Prevalence of Neutralizing Antibodies to the Common Adenoviruses in Children With Cystic Fibrosis: Implication for Gene Therapy With Adenovirus Vectors

Incidence and Prevalence of Neutralizing Antibodies to the Common Adenoviruses in Children With Cystic Fibrosis: Implication for Gene Therapy With Adenovirus Vectors

infection. Limited published data on serum neutral- izing antibody in healthy children are available, but to the best of our knowledge no study in CF children has been performed. In our study, CF children seemed to make a normal serum neutralizing anti- body response to Ad infection. The level of neutral- izing antibody titers to the Ads in seropositive CF children was similar to those in adults and persisted for at least a year. An age-specific increase in anti- body prevalence was observed to the common Ads. By defining the prevalence of antibodies to the com- mon Ads based on neutralizing antibody, a rational for sequential administration of replication-deficient recombinant Ad vectors of different serotypes may be developed for gene therapy to CF children. This concept may prove to be of importance if the admin- istration of the Ad vector in the lungs of CF children with preexisting serum neutralizing antibody spe- cific to the Ad vector interferes with the infection by the vector of target cells and the subsequent transfer of the CFTR gene.
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Gene therapy strategies for treating brain tumors: Retroviruses are still good candidates for therapeutic vectors

Gene therapy strategies for treating brain tumors: Retroviruses are still good candidates for therapeutic vectors

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In the past few decades, many efforts have been made to im- prove the prognosis of GBM, however, with limited success. Many gene therapy strategies for GBM have been developed and a few have progressed to clinical trials. Retroviral vectors have superior features for gene therapy in brain cancers, including tumor speci- ficity, immunogenicity, and longer half-life. Early gene therapy trials in GBM patients based on transplanta- tion of retrovirus-producing cells into the brain failed to prove efficacious. Adenoviral vectors, which can be prepared as high-titer virus solutions and undergo ef- ficient transduction in tumor cells, failed in clinical trials, likely due to immunogenicity and instability of gene expression. Alternative therapeutics such as on- colytic viruses that specifically target and destroy can- cer cells are currently under investigation. In addi- tion to novel vectors, retroviral vectors are still at- tractive candidates for use in gene therapy against brain tumors. Since yields of properly-packaged viral particles from virus-producing cells have been very li- mited so far, gene therapy by direct injection of high- titer retroviral vectors into the patients’ brains was not possible. To overcome these disadvantages, a pa- ckaging cell line that yields high-titer retroviral solu- tions was established by our group, enabling the di- rect injection of massive retroviral vector stocks di- rectly into the brain. Mouse glioma models were ef- fectively cured with a combination of a suicide gene/ prodrug system and a highly-concentrated retrovirus solution. Preclinical assessments, including that of re- plication-competent retroviruses and tumorigenicity of the combination method, have confirmed the safety of the highly-concentrated retrovirus solution. Addi-
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Adenovirus Vectors with the 100K Gene Deleted and Their Potential for Multiple Gene Therapy Applications

Adenovirus Vectors with the 100K Gene Deleted and Their Potential for Multiple Gene Therapy Applications

The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K ⴚ ]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1 ⴚ ,100K ⴚ ]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K ⴚ ]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K ⴚ ]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1 ⴚ ,100K ⴚ ]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K ⴚ ]Ad vectors suggest that they may have utility in a variety of gene therapy applications.
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Self-deleting retrovirus vectors for gene therapy.

Self-deleting retrovirus vectors for gene therapy.

Gene therapy, one of the fastest-growing areas in medical research, bears the promise that in the near future it may be possible to correct aberrant gene function by the expression of normal genes in somatic cells. Although several viral and non- viral vectors have been employed to deliver genes to mamma- lian cells, the most widely used are based on Moloney murine leukemia virus (MoMoLV), a retrovirus that normally infects mice (reviewed in references 7, 22, 26, and 28). These vectors are easy to make and are the most efficient agents yet identified for transfer of genes into human cells. For these reasons, a crippled version of the MoMuLV retrovirus has been used in more than 75% of the human trials approved to date (7). However, like all of the other vectors considered for gene delivery into human cells, retroviruses have inherent problems. First, viral vector sequences can recombine with endogenous or exogenous helper viruses to generate new and unpredictable forms of infectious virus (9, 38; reviewed in reference 36). Second, rates of transfer and expression vary dramatically be- tween different cells and different patients. This may result from integration- or cell type-specific events, including gene inactivation by methylation (5), or by transcriptional repressors that bind the viral genome (2, 5, 15, 46). Third, retroviruses integrate mostly randomly throughout the genome, thus in- creasing the risk of cancer when activating insertions occur alongside cellular oncogenes (9, 38; reviewed in reference 39). Fourth, selectable marker or reporter genes commonly used in ex vivo gene therapy often interfere with the metabolism of the recipient cells (14, 37, 42) and may also trigger an immune response (reviewed in reference 25). Finally, most retroviruses require cell division for gene insertion, thus precluding their use for nondividing targets (24).
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Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardiza- tion of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy.
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THE USE OF VIRAL VECTORS IN GENE TRANSFER THERAPY

THE USE OF VIRAL VECTORS IN GENE TRANSFER THERAPY

Gene therapy is strategy based on using genes as pharmaceuticals. Gene therapy is a treatment that involves altering the genes inside body's cells to stop disease. Genes contain DNA- the code controlling body form and function. Genes that do not work properly can cause disease. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve the ability of the body to fight disease. Gene therapy holds promise for treating a wide range of diseases, including cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Various types of genetic material are used in gene therapy; double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA and antisense oligodeoxynucleotides (ASON). The success of gene therapy depends on assuring the entrance of the therapeutic gene to targeted cells without any form of biodegradation. Commonly used vectors in gene therapy are: adenoviruses (400 clinical studies; 23.8%), retroviruses (344 clinical studies; 20.5%), unenveloped/plasmid DNA (304 clinical studies, 17.7%), adeno- associated viruses (75 clinical studies; 4.5%) and others. In this paper, we have reviewed the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors.
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