RESUMO
We report the safety and tolerability of 87 infusions of lentiviral vectormodified 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 â 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 vectortransduced 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.
Assuntos
Linfócitos T CD4-Positivos/transplante , Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1/genética , Lentivirus/genética , Oligonucleotídeos Antissenso/farmacologia , Transferência Adotiva/métodos , Adulto , Antivirais/efeitos adversos , Antivirais/metabolismo , Antivirais/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Feminino , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Vetores Genéticos/metabolismo , Vetores Genéticos/farmacologia , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Lentivirus/metabolismo , Lentivirus/fisiologia , Masculino , Pessoa de Meia-Idade , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/efeitos adversos , Oligonucleotídeos Antissenso/genética , Transdução Genética/métodos , Carga Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
γ-Retroviral vectors (γ-RV) are powerful tools for gene therapy applications. Current clinical vectors are produced from stable producer cell lines which require minimal further downstream processing, while purification schemes for γ-RV produced by transient transfection have not been thoroughly investigated. We aimed to develop a method to purify transiently produced γ-RV for early clinical studies. Here, we report a simple one-step purification method by high-speed centrifugation for γ-RV produced by transient transfection for clinical application. High-speed centrifugation enabled the concentration of viral titers in the range of 107-108 TU/mL with >80% overall recovery. Analysis of research-grade concentrated vector revealed sufficient reduction in product- and process-related impurities. Furthermore, product characterization of clinical-grade γ-RV by BioReliance demonstrated two-logs lower impurities per transducing unit compared with regulatory authority-approved stable producer cell line vector for clinical application. In terms of CAR T cell manufacturing, clinical-grade γ-RV produced by transient transfection and purified by high-speed centrifugation was similar to γ-RV produced from a clinical-grade stable producer cell line. This method will be of value for studies using γ-RV to bridge vector supply between early- and late-stage clinical trials.
RESUMO
In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.
Assuntos
Genes env , Vetores Genéticos , Lentivirus/genética , Oligonucleotídeos Antissenso/farmacologia , Vírus da Imunodeficiência Símia/genética , Replicação Viral/efeitos dos fármacos , Animais , Antígenos CD34/metabolismo , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Genes env/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos T/metabolismo , Transdução GenéticaRESUMO
This review is intended to exemplify the roles and responsibilities of the two agencies under the Department of Health and Human Services, the National Institutes of Health and the Food and Drug Administration, that have oversight for human gene transfer clinical protocols, as seen through our experience of bringing a first-in-its-class lentiviral vector to clinical trials. In response to the changing circumstances in gene therapy research between 1999 and 2002, the concerns of these agencies regarding gene therapy have been evolving. This review provides an overview of the major safety concerns regarding insertional oncogenesis, the generation of a replication- competent lentivirus (RCL), and vector mobilization thought to be related to lentiviral vectors, which had to be addressed during the regulatory review process before initiating the clinical trial. Specific monitoring assays to address these concerns were established to test for RCL generation, vector mobilization, persistence of vector-modified cells, and abnormal clonal expansion of modified cells. We hope to provide a basic understanding and appreciation of the regulatory process and major safety concerns, toward providing useful insight to those presently embarking on the development of clinical application of lentiviral vectors.
Assuntos
Terapia Genética , Vetores Genéticos , Lentivirus/genética , Neoplasias/terapia , Pesquisa Biomédica/legislação & jurisprudência , Ensaios Clínicos como Assunto , Regulamentação Governamental , Experimentação Humana/normas , Humanos , Neoplasias/genéticaRESUMO
The intracellular activity of certain antiviral agents, including antisense oligonucleotides, acyclic nucleoside phosphonates, and protease inhibitors, is enhanced when they are delivered in liposome-encapsulated form. In this chapter we describe the preparation of pH-sensitive liposomes encapsulating antisense oligonucleotides, ribozymes, and acyclic nucleoside phosphonate analogues and their effects on HIV replication in macrophages. We outline the use of liposomal HIV protease inhibitors in infected macrophages. We present two methods for the covalent coupling of soluble CD4 to liposomes and show the association of these liposomes with HIV-infected cells. We also describe the synthesis of a novel antiviral agent based on cyclodextrin and its incorporation into liposomes.
Assuntos
Antivirais/administração & dosagem , Sistemas de Liberação de Medicamentos , Lipossomos , Antivirais/metabolismo , Antivirais/uso terapêutico , Antígenos CD4/química , Antígenos CD4/metabolismo , Reagentes de Ligações Cruzadas/química , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lipossomos/química , Lipossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Testes de Sensibilidade Microbiana , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Organofosfonatos/química , Organofosfonatos/uso terapêutico , Inibidores de Proteases/metabolismo , Inibidores de Proteases/uso terapêutico , Purinas/química , Purinas/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , RNA Catalítico/metabolismo , RNA Catalítico/uso terapêutico , Succinimidas/química , Sulfetos/química , beta-Ciclodextrinas/química , beta-Ciclodextrinas/metabolismoAssuntos
Concentração de Íons de Hidrogênio , Lipossomos/química , Lipossomos/síntese química , Animais , Portadores de Fármacos , Estabilidade de Medicamentos , Fluoresceínas , Lipossomos/farmacocinética , Microscopia de Fluorescência/métodos , Oligodesoxirribonucleotídeos Antissenso/farmacocinética , Ratos , Rodaminas , EstereoisomerismoRESUMO
We report findings from a clinical evaluation of lentiviral vectors in a phase I open-label nonrandomized clinical trial for HIV. This trial evaluated the safety of a conditionally replicating HIV-1-derived vector expressing an antisense gene against the HIV envelope. Five subjects with chronic HIV infection who had failed to respond to at least two antiviral regimens were enrolled. A single i.v. infusion of gene-modified autologous CD4 T cells was well tolerated in all patients. Viral loads were stable, and one subject exhibited a sustained decrease in viral load. CD4 counts remained steady or increased in four subjects, and sustained gene transfer was observed. Self-limiting mobilization of the vector was observed in four of five patients. There is no evidence for insertional mutagenesis after 21-36 months of observation. Immune function improved in four subjects. Lentiviral vectors appear promising for gene transfer to humans.
Assuntos
Vetores Genéticos/genética , Lentivirus/fisiologia , Replicação Viral/genética , Adulto , Técnicas de Transferência de Genes , HIV-1/genética , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Pessoa de Meia-Idade , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
BACKGROUND: A stable packaging cell line facilitates large-scale lentivirus vector manufacture. However, it has been difficult to produce clinical-scale HIV-1-based lentiviral vectors using a packaging cell line, in part due to toxicity of packaging genes, and gene silencing that occurs during the long culture period necessary for sequential addition of packaging constructs. METHODS: To avoid these problems, we developed a three-level cascade gene regulation system designed to remove tetracycline transactivator (tTA) from cytomegalovirus immediate early promoter (CMV)-controlled expression to reduce cytotoxicity from constitutive expression of tTA and leaky expression of packaging genes. We also performed a one-step integration of the three packaging plasmids to shorten the culture time for clonal selection. RESULTS: Although leaky expression of p24 and vector production still occurred despite the three-level regulation system, little cytotoxicity was observed and producer cells could be expanded for large-scale production. Producer cells yielded remarkably stable vector production over a period greater than 11 days with the highest titer 3.5 x 10(7) transducing units (TU)/ml and p24 300 ng/ml, yielding 2.2 x 10(11) TU and 1.8 milligram (mg) p24 from one cell factory. No replication-competent lentivirus (RCL) was detected. Long-term analysis demonstrated that, although the cells are genetically stable, partial gene silencing occurs after 2-3 months in culture; however, the one-step construct integration allowed prolonged vector production before significant gene silencing. Concentrated vector resulted in 90% transduction in CD4+ lymphocytes at 20 TU per cell. CD34+ progenitor cells were transduced at 41-46% efficiency, and long-term initiating culture (LTC-IC) was transduced at 45-51%. CONCLUSIONS: These results demonstrate for the first time HIV-1-based lentiviral vector production on the large scale using a packaging cell line.
Assuntos
Vetores Genéticos/genética , HIV-1/genética , Lentivirus/genética , Sequência de Bases , Linhagem Celular , Células Clonais , Clonagem Molecular , Códon , Ensaio de Imunoadsorção Enzimática , Proteínas de Fusão gag-pol/química , Proteínas de Fusão gag-pol/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Produtos do Gene rev/química , Produtos do Gene rev/genética , Produtos do Gene tat/química , Produtos do Gene tat/genética , Engenharia Genética , Vetores Genéticos/biossíntese , Células HeLa , Humanos , Cinética , Glicoproteínas de Membrana/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos , Tetraciclina/farmacologia , Transdução Genética , Transfecção , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Replicação Viral , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34(+) bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34(+) cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4(+) GFP(+) T cells derived from VRX494-transduced CD34(+) cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS.
Assuntos
Antígenos CD34/biossíntese , Linfócitos T CD4-Positivos/imunologia , HIV-1/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Vírus da Imunodeficiência Símia/genética , Replicação Viral , Adenosina Desaminase/metabolismo , Animais , Antígenos CD34/genética , Southern Blotting , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Citometria de Fluxo , Produtos do Gene env/metabolismo , Produtos do Gene rev/metabolismo , Produtos do Gene tat/metabolismo , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/virologia , Humanos , Leucócitos Mononucleares/metabolismo , Macaca mulatta , Modelos Genéticos , Oligonucleotídeos Antissenso/química , RNA/química , Retroviridae/genética , Células-Tronco/metabolismo , Linfócitos T/metabolismo , Regulação para Cima , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
We report the design of a unique two-plasmid production system for the first lentiviral vector to be evaluated in humans, VRX496. VRX496 is an optimized VSV-G pseudotyped vector derived from HIV-1 that expresses antisense to the HIV envelope gene. We found that a two-plasmid approach to production resulted in higher vector production titers when compared with a three-plasmid approach, which is particularly important for vector production at the large scale. Therefore, we carefully designed a single packaging construct, VIRPAC, for safety by reducing its homology with VRX496 and by insertion of functionally validated genetic elements designed to reduce the risk of generation of a replication-competent lentivirus (RCL). A native cis-acting ribozyme is used to prevent read through into the envelope gene from the upstream gag-pol genes in the packaging vector, thus preventing RNAs containing gag-pol and env together for comparable safety to a three-plasmid system. We demonstrate that there is no significant in vivo vector mobilization using a primary SCID-hu mouse transplantation model, which correlates with the presence of an anti-HIV payload and suggests that inclusion of antisense may be a useful tool to restrict mobilization in other vector constructs. Gene transfer is achieved using a one-step transduction procedure that is simple and clinically translatable, which reaches stable transduction efficiencies of >99% in CD4+ T lymphocytes within 3 days of culture initiation.
Assuntos
Terapia Genética , Vetores Genéticos/genética , Lentivirus/genética , Plasmídeos/genética , Animais , Elementos Antissenso (Genética)/genética , Células Cultivadas , Produtos do Gene env/genética , Engenharia Genética , Vetores Genéticos/efeitos adversos , Vetores Genéticos/biossíntese , HIV-1/genética , Humanos , Camundongos , Camundongos SCID , RNA Catalítico/genética , RNA Catalítico/fisiologia , RNA Viral/metabolismo , Linfócitos T , Transdução Genética , Proteínas Virais/genéticaRESUMO
High gene transfer efficiencies have been difficult to achieve in hematopoietic progenitor cells (HPCs) but are important to therapeutic success of HPC gene therapy. Efficient gene transfer is especially challenging with use of column-purified vector for clinical application, as opposed to centrifuged vector commonly used for research. We investigated novel approaches to increase transduction by using a clinically applicable protocol and quantities of column-purified lentiviral vector. Recognizing the association of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters with HPC biology, we investigated the effect of transporter inhibitors on transduction. We found the ABC transporter inhibitor verapamil improved transduction efficiency 2- to 6-fold into CD34(+) cells isolated from mobilized peripheral blood, bone marrow, and cord blood. Verapamil also improved transduction in human SCID (severe combined immunodeficient) repopulating cell (SRC) transduction 3- to 4-fold, resulting in 80% to 90% transduction levels in mice receiving primary and secondary transplants without alterations in multilineage reconstitution. Additional ABC transporter substrate inhibitors like quinidine, diltiazem, and ritonavir also enhanced transduction 2- to 3-fold, although ABC transporter inhibitors that are not substrates did not. Enhanced transduction was not observed in mature hematopoietic cells, neurospheres, mesenchymal stem cells, or hepatocytes. Enhancement of transduction in HPCs was observed with vesicular stomatitis virus-G (VSV-G)-pseudotyped lentiviral vector but not with vector pseudotyped with RD114. Thus, we present a new approach for efficient delivery to primitive HPCs by VSV-G-pseudotyped lentiviral vectors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Vetores Genéticos , Células-Tronco Hematopoéticas/fisiologia , Lentivirus/genética , Transdução Genética/métodos , Adulto , Animais , Antígenos CD34/análise , Bloqueadores dos Canais de Cálcio/farmacologia , Centrifugação , Diltiazem/farmacologia , Terapia Genética/métodos , Células HeLa , Células-Tronco Hematopoéticas/química , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Quinidina/farmacologia , Verapamil/farmacologiaRESUMO
We present preclinical studies that demonstrate in vitro the feasibility and efficacy of lentivirus-based vector antisense gene therapy for control of HIV replication in primary T lymphocytes isolated from HIV-infected patients discordant for clinical status. VRX496 is a VSV-G-pseudotyped HIV-based vector that encodes an antisense payload against the HIV envelope gene. The antisense payload is under the control of the native LTR promoter, which is highly transactivated by tat upon HIV infection in the cell. Transfer of autologous CD4(+) T lymphocytes genetically modified with VRX496 (VRX496T) into HIV-infected patients is intended to provide a reservoir of cells capable of controlling HIV, potentially delaying AIDS onset. To determine the patient population likely to respond to VRX496 for optimal efficacy, we examined the ability of our research vector, VRX494, to modify and suppress HIV in vitro in lymphocytes isolated from 20 study subjects discordant for CD4 count and viral load. VRX494 is analogous to the clinical vector VRX496, except that it contains GFP as a marker gene instead of the 186-tag marker in the clinical vector. To transfer VRX494 to target cells we developed a novel scalable two-step transduction procedure that has been translated to the clinic in an ongoing clinical trial. This procedure achieved unprecedented transduction efficiencies of 94 +/- 5% in HIV(+) study subject cells. In addition the vector inhibited HIV replication >/=93% in culture regardless of the viral load or CD4 count of the subject or tropism of the virus strain with which they were infected. These findings demonstrate that VRX496T therapy is expected to be beneficial to patients that differ in their status in term of CD4 count and viral load. The methods described represent significant technical advances facilitating execution of lentivirus vector-mediated gene therapy for treatment of HIV and are currently being employed in the first trial evaluating lentivirus vector safety in humans.