Perry Hall, MD, United States
Perry Hall, MD, United States

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McGarrity G.J.,VIRxSYS Corporation | Hoyah G.,VIRxSYS Corporation | Winemiller A.,VIRxSYS Corporation | Winemiller A.,U.S. National Institutes of Health | And 12 more authors.
Journal of Gene Medicine | Year: 2013

Background: Lentiviral vectors are being used with increasing frequency in human clinical trials. We were the first to use lentiviral vectors in clinical trials in 2003. Our lentiviral vector encoded a long RNA antisense sequence to the HIV-1 envelope and was used in an ex vivo autologous setting to provide viral load control in HIV-1 positive subjects failing anti-HIV therapy. A total of 65 subjects have been treated in Phase 1 and Phase 2 trials in six institutions. Methods: Good manufacturing practices (GMP) lots of the lentiviral vector used in our clinical trials were assayed for the presence of replication competent lentivirus (RCL). RCL assays were conducted at two stages. The first testing was performed on samples collected immediately following bulk harvest of the GMP product lot and consisted of 1×108 cells used in production. RCL assays were also performed on aliquots of the final fill of the vector by the inoculation of at least 5% of the GMP final fill volume into C8166 cells, passaged for at least ten passages and tested for RCL by p24 enzyme-linked immunosorbent assay and vesicular stomatitis virus-G envelope DNA. Results: Following 263 infusions of autologous, transduced cells, no adverse events have been detected in these subjects, with some followed for more than 8years following infusions. More than 4.3×1012 VRX496 proviral copies were administered to these 65 subjects. Conclusions: Data from this small population suggest that there is no apparent risk for serious adverse events with the use of lentiviral vectors. © 2013 John Wiley & Sons, Ltd.

Tebas P.,University of Pennsylvania | Stein D.,Jacobi Medical Center | Binder-Scholl G.,University of Pennsylvania | Mukherjee R.,University of Pennsylvania | And 24 more authors.
Blood | Year: 2013

We report the safety and tolerability of 87 infusions of lentiviral vector-modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vector-transduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells. This study is registered at as number NCT00295477. © 2013 by The American Society of Hematology.

Asefa B.,VIRxSYS Corporation | Korokhov N.,VIRxSYS Corporation | Lemiale F.,VIRxSYS Corporation
Vaccine | Year: 2010

Viral vectors are considered as one of the major means for the induction of strong immune responses against recombinant antigens by genetic immunization. Among these, lentiviral vectors are particularly attractive vehicles, as they can infect a wide variety of cells and can transduce replicating as well as non-replicating cells. We have engineered VRX1023, an HIV-1-based lentiviral vector (LV) vaccine candidate, to deliver HIV-1 Gag, Pol and Rev antigens under control of the native LTR promoter. While VRX1023 has been shown to elicit strong cell-mediated and humoral immunity as a stand-alone vaccine, we report here its combination in a heterologous prime-boost approach. Its combination with an adenovirus serotype 5 (Ad5)-based vector in the mouse model increased the frequency and polyfunctionality of HIV-specific CD4+ and CD8+ T cells. Homologous prime-boost regimens induced high levels of anti-vector neutralizing antibodies in Ad5-immunized mice, whereas the VSV-G-pseudotyped VRX1023 LV elicited low levels of anti-lentiviral vector neutralization. In addition, the heterologous prime-boost strategy resulted in a 5-fold reduction in Ad5-specific vector neutralization as compared to Ad5 homologous immunization. In conclusion, this study demonstrates that LV and Ad5 vector candidates can be combined in a heterologous immunization regimen, yielding dramatically improved immunogenicity while overcoming anti-vector immunity. These findings may have implications for the development of HIV vaccine regimens in populations with elevated Ad5 seroprevalence or when repeated vector administrations are required. © 2010 Elsevier Ltd. All rights reserved.

Lemiale F.,VIRxSYS Corporation | Asefa B.,VIRxSYS Corporation | Ye D.,VIRxSYS Corporation | Chen C.,VIRxSYS Corporation | And 2 more authors.
Vaccine | Year: 2010

Recently developed viral-vectored HIV vaccine candidates, despite achieving high levels transgene expression and inducing high magnitude immune responses to HIV, have faced limitations related to anti-vector immunity. In contrast, lentiviral vectors (LV) have been shown to be less sensitive to anti-vector neutralizing activity, while displaying desirable characteristics, such as transduction of non-dividing cells, including antigen-presenting cells, and long-term transgene expression. We have developed VRX1023, an HIV-based LV expressing HIV Gag, Pol and Rev under the control of the native HIV LTR. In mice, this vector induced significant mucosal and systemic cellular and humoral responses against HIV after sub-cutaneous injection. Similarly to other viral vectors, this LV candidate can be effectively used in DNA prime, LV boost strategies, where it elicited as high as 21% HIV Gag-specific CD8 responses as measured by intracellular cytokine staining. Moreover, anti-vector immunity is not an obstacle to repeated LV administrations, as shown by improved anti-HIV responses compared to single LV immunization. In head to head comparisons with Ad5 vectors expressing the same vaccine payload, VRX1023 elicited higher and more persistent cellular and antibody responses to HIV than its adenoviral counterpart. In preparation for clinical use, manufacturing scale-up of a highly purified VRX1023 vector lot following cGMP was successfully achieved without altering the robust immunogenicity observed with the research-grade vector. VRX1023, in addition to competing favorably with existing vectors such as Ad5 for anti-HIV immune responses, demonstrates unique features likely to address some of the pitfalls of current vector-based HIV vaccine strategies. © 2009 Elsevier Ltd. All rights reserved.

Kimpel J.,Chemotherapeutisches Forschungsinstitut Georg Speyer Haus | Braun S.E.,Harvard University | Qiu G.,Harvard University | Wong F.E.,Harvard University | And 12 more authors.
PLoS ONE | Year: 2010

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that. 95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes. © 2010 Kimpel et al.

Willerth S.M.,University of California at Berkeley | Pedro H.A.M.,University of California at Berkeley | Pachter L.,University of California at Berkeley | Humeau L.M.,VIRxSYS Corporation | And 2 more authors.
PLoS ONE | Year: 2010

Background: With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies. Methodology/Principal Findings: We report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patient's infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity. Significance: This study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure. © 2010 Willerth et al.

Virxsys Corporation | Date: 2012-08-10

Methods and compositions are provided for generating novel nucleic acid molecules through targeted spliceosome mediated RNA trans-splicing (SMaRT) that result in expression of a splicing isoform or variant thereof. The methods and compositions are based upon pre-trans-splicing molecules (PTMs) designed to interact with a target pre-mRNA molecule and mediate a trans-splicing reaction generating a novel chimeric RNA molecule encoding a splicing isoform for the treatment of a variety of gene isoform induced diseases such as cancer.

The present invention provides methods and compositions for generating novel nucleic acid molecules through RNA trans-splicing that target a highly expressed pre-mRNA and contain the coding sequence for antibody polypeptide(s). The compositions of the invention include pre-trans-splicing molecules (PTMs) designed to interact with the target precursor messenger RNA molecule (target pre-mRNA) that is abundantly expressed or tumor specific and mediate a trans-splicing reaction resulting in the generation of novel chimeric RNA molecule (chimeric RNA) capable of encoding an antibody polypeptide. The invention provides for the in vivo production of chimeric RNA molecules that encode and result in the production of an antibody polypeptide that is therapeutically effective against, for example, infectious agents, cancer cells, transplantation antigens, rheumatoid arthritis, etc.

Virxsys Corporation and Intronn Inc. | Date: 2011-12-06

Biochemicals used in the science and technology industry, namely, precursors for in vitro genetic engineering use, precursors used for creating genetically engineered plants, precursors used for creating generically engineered agricultural crops, and precursors used as diagnostic preparations and reagents for scientific or research use; reagents for scientific or medical research use; kits consisting primarily of RNA molecules, pre-trans-splicing molecules, cultured cells and molecular probes, for research in laboratories, namely, biotechnology related research and genetic engineering research. Pharmaceutical preparations for medical uses, namely, for the treatment of genetic diseases, cancer, and infections with pathogenic organisms; reagents for medical diagnostics; vaccines for the treatment of both animal and human diseases including bacterial and viral diseases; biochemicals for use in the treatment of cardiovascular disease, hemophilia, cancer, and diseases linked to or caused by one or more genes; Kits for diagnosing pathological or genetic disorders consisting primarily of one or more of the following - pre-trans-splicing molecules plasmid DNA; lentiviral vector containing pre-trans-splicing molecules; pre-trans-splicing molecules with fluorescent dyes attached; buffer for the reactions; molecular biology reagents; genes within pre-trans-splicing molecules for fluorescent probes. Scientific research and product research, namely, research on genomes. Medical services and medical testing, namely, diagnostics and prognostics; agricultural advice.

Methods and compositions for generating novel nucleic acid molecules through targeted spliceosome mediated RNA trans-splicing that result in expression of a apoAI protein, an apoAI variant, the preferred embodiment referred to herein as the apoAI Milano variant, a pre-pro-apoAI or an analogue of apoAI. The methods and compositions include pre-trans-splicing molecules (PTMs) designed to interact with a target precursor messenger RNA molecule (target pre-mRNA) and mediate a trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (chimeric RNA) capable of encoding apoAI, the apoAI Milano variant, or an analogue of apoAI. The expression of this apoAI protein results in protection against vascular disorders resulting from plaque build up, i.e., atherosclerosis, strokes and heart attacks.

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