RESUMEN
In a phase 1 clinical trial, we are evaluating a murine leukemia virus (MuLV)-based retroviral vector encoding the human factor VIII gene [hFVIII(V)], administered intravenously, as a therapy for hemophilia A. Preclinical biolocalization studies in adult rabbits revealed vector-specific PCR signals in testis tissue at low levels. In follow-up animal studies we used PCR to (1) estimate the frequency with which a given cell in testis tissue is transduced, and (2) determine whether a positive PCR signal could be detected in semen samples from animals treated with hFVIII(V). Using the 99% confidence bound, results indicate that the probability that a given cell within the testis was transduced is less than 1/709,000 (97 days after treatment). This probability decreased with time after hFVIII(V) administration. Moreover, the rate of provector sequence detection in semen samples collected weekly throughout two cycles of spermatogenesis was 3/4281 reactions (0.07%), which is lower than the rate of false positives (1/800, 0.125%) observed for control animals. Using PCR assays with single-copy sensitivity, we have shown that the small number of transduced cells present in testis tissue does not give rise to detectable transduced cells in semen.
Asunto(s)
Factor VIII/genética , Retroviridae/genética , Semen/metabolismo , Testículo/metabolismo , Animales , Vectores Genéticos , Masculino , Modelos Biológicos , Modelos Estadísticos , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa , Conejos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espermatogénesis , Factores de Tiempo , Distribución Tisular , Transducción GenéticaRESUMEN
For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.
Asunto(s)
Vectores Genéticos , Retroviridae/genética , Ensamble de Virus , Secuencia de Bases , Línea Celular , Cartilla de ADN , Factor VIII/genética , Hemofilia A/terapia , HumanosRESUMEN
Gene delivery via murine-based recombinant retroviral vectors is currently widely used in gene therapy clinical trials. The vectors are engineered to be replication defective by replacing the structural and nonstructural genes of a cloned infectious retrovirus with a therapeutic gene of interest. The retroviral particles are currently generated in packaging cell lines, which supply all retroviral proteins in trans. Recombination between short homologous regions of the retroviral vector and packaging cell line elements can theoretically generate replication-competent retrovirus (RCR) and hence the Food and Drug Administration (FDA) requires the monitoring of clinical trial subjects for the presence of RCR. Sensitive polymerase chain reaction (PCR) assays have been used for the detection of murine leukemia virus (MLV) nucleotide sequences in peripheral blood mononuclear cells (PBMCs). A novel serological enzyme-linked immunosorbent assay (ELISA) for the detection of anti-MLV specific immunoglobulin (Ig) has been developed to be used as an alternative to the PCR assay. Both assays were used to monitor human immunodeficiency virus (HIV)-positive clinical trial subjects who had received multiple injections of HIV-IT (V), a retroviral vector encoding HIV-1 IIIBenv/rev. Western blot analysis and an in vitro vector neutralization assay were used to characterize further a subset of serum samples tested by ELISA. Results show no evidence of RCR infection in clinical trial subjects. PCR and ELISA assays are discussed in terms of their advantages and limitations as routine screening assays for RCR. The PCR assay is our current choice for monitoring clinical trial subjects receiving direct administration of vector, and the ELISA is our choice for those receiving ex vivo treatment regimens.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Vectores Genéticos/uso terapéutico , Reacción en Cadena de la Polimerasa/métodos , Retroviridae/genética , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Ensayos Clínicos Fase I como Asunto , Estudios de Evaluación como Asunto , Vectores Genéticos/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/virología , Humanos , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/inmunología , Replicación ViralRESUMEN
Replication-incompetent retroviruses have been employed as gene therapy vectors in experimental settings for more than a decade. More recently, these vectors have been tested in the clinic as immunotherapeutic agents and anticancer agents. One potential problem with the use of such vectors is the possible development of immune responses directed against the vector particles themselves. Here, we examine immunoglobulin (Ig) responses specific for retroviral vectors derived from murine leukemia virus (MLV). Anti-MLV Ig is seen following intramuscular (i.m.) administration of retroviral vectors in mice, and in nonhuman primates; as expected, these responses are dependent upon the vector dose delivered. Furthermore, serum from vector-treated animals is capable of partially neutralizing vector-mediated transduction of target cells in an in vitro assay. Nevertheless, even in the presence of significant levels of anti-vector Ig in vivo, i.m. administration of retroviral vector is still capable of driving both Ig and cytotoxic T lymphocyte (CTL) responses specific for vector-encoded gene products. This work suggests that although retroviral vectors may readily induce immune responses directed against the vector particles themselves, such responses will not significantly affect the efficiency of these vectors in an immunotherapeutic protocol.
Asunto(s)
Vectores Genéticos/inmunología , Vectores Genéticos/farmacología , Inmunoglobulinas/efectos de los fármacos , Inmunoglobulinas/inmunología , Retroviridae/genética , Animales , Relación Dosis-Respuesta a Droga , Femenino , Vectores Genéticos/genética , Immunoblotting , Inmunoglobulina G/efectos de los fármacos , Inmunoglobulina G/metabolismo , Virus de la Leucemia Murina/genética , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Primates , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Retroviridae/inmunología , beta-Galactosidasa/genética , beta-Galactosidasa/inmunología , beta-Galactosidasa/metabolismoRESUMEN
We have developed a novel gene transfer drug, HIV-IT(V), for the treatment of HIV infection in humans. HIV-IT(V) is a retroviral vector encoding the HIV-1 IIIB env and rev genes and a neomycin resistance marker gene (neor). We have recently reported that HIV-IT(V) administered intramuscularly to male mice localizes primarily to the site of injection. In this study, we have investigated more extensively the localization and biological activity of HIV-IT(V) administered intramuscularly to female mice. Consistent with our previous findings, retroviral DNA was detected by PCR at the site of injection. Retroviral DNA was also detected in proximal lymph nodes, a tissue not examined previously. Potential for drainage of vector particles to regional lymph nodes was indicated by experiments showing that intramuscular injection of fluorescein-labeled latex beads concentrated in the regional lymph nodes in mice. The localization of retroviral DNA to the injection site and regional lymph nodes may play a role in the induction of the HIV-specific CTL responses detected in splenocyte populations isolated from mice 21 days after injection with HIV-IT(V).
Asunto(s)
ADN Viral/análisis , Vectores Genéticos/administración & dosificación , VIH-1/genética , Virus de la Leucemia Murina/genética , Ganglios Linfáticos/virología , Animales , Secuencia de Bases , ADN Recombinante/análisis , Femenino , Técnicas de Transferencia de Gen , Genes env/genética , VIH-1/inmunología , Inyecciones Intramusculares , Ganglios Linfáticos/química , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Músculo Esquelético/química , Reacción en Cadena de la Polimerasa/métodos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Many protocols for gene therapy employ recombinant retroviral vectors, which are replication-defective retroviruses engineered to serve as gene delivery vehicles. The use of retroviral vectors for human gene therapy requires careful screening of vector-producing cell lines and culture supernatants to ensure the absence of replication competent retrovirus (RCR) in clinical products. In this study we have examined several different culture assays routinely used to test for the presence of RCR. Results indicate that cocultivation of a vector-producing cell line with a permissive cell line can reproducibly detect a low level of contaminating RCR. RCR was detected less frequently in direct tests of cell-free culture supernatants from a contaminated vector-producing line. Further studies revealed that recombinant retroviral vector can interfere, to varying degrees, with the detection of low-level RCR in culture supernatants when a marker rescue assay, an extended mink S+L- assay or a PG-4 S+L- assay is used. Interference can be partially overcome by culturing the vector preparation with a permissive cell line for several days before testing on the indicator cell line. The interference phenomenon we have observed may also occur in other culture assays routinely used for the detection of RCR.
Asunto(s)
Artefactos , Bioensayo , Línea Celular/virología , Medios de Cultivo Condicionados/análisis , Virus Defectuosos/aislamiento & purificación , Terapia Genética/normas , Vectores Genéticos/aislamiento & purificación , Retroviridae/aislamiento & purificación , Retroviridae/fisiología , Ensayo de Placa Viral , Replicación Viral , Animales , Técnicas de Cultivo/métodos , Perros , Reacciones Falso Negativas , Prueba de Complementación Genética , Marcadores Genéticos , Vectores Genéticos/normas , Visón , Muridae , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Recombinación Genética , Reproducibilidad de los Resultados , Seguridad , Sensibilidad y EspecificidadRESUMEN
A murine retroviral vector encoding the human immunodeficiency virus type 1 (HIV-1) env and rev genes can be used to induce cytotoxic T lymphocyte responses. Immune responses can be induced by an ex vivo treatment, in which autologous cells are transduced in vitro and re-introduced to the donor, or by direct administration of retroviral vector via intramuscular injection. In this study we have used polymerase chain reaction (PCR) analysis to examine the distribution of recombinant murine retrovirus directly administered to mice. Mice were injected intramuscularly with HIV-IT(V), an amphotropic murine leukemia virus (MLV)-based retroviral vector carrying the HIV-1 env/rev genes and a neomycin resistance marker gene. Detection of the HIV-1 env gene in DNA isolated from injection sites demonstrated in vivo transduction. No evidence of transduction was observed in the testes, spleen, kidney, or thymus. Retroviral DNA was detected in the liver of one animal in the study. These data suggest that retroviral vector administered intramuscularly to mice localizes primarily to the site of injection and that measurable transduction in the testes does not occur.