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
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
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
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.