Biodistribution and Toxicity Studies of VSVG-Pseudotyped Lentiviral Vector after Intravenous Administration in Mice with the Observation of in Vivo Transduction of Bone Marrow

Abstract

Lentiviral vectors can confer high levels of gene transfer and transgene expression in a variety of cell types. However, the biodistribution and toxicity after intravenous administration have not been reported. To address these issues of biodistribution and toxicity, an HIV-1-based vector, HR'cmvGFP, was administered to normal BALB/c mice by tail-vein injection. Nine different organs and bone marrow were evaluated by real-time quantitative PCR (QPCR) assay capable of a broad range of quantitation (5-log fold) to detect as few as one copy of the green fluorescent protein gene (GFP) per 10(5) cells. Four days after vector administration, high levels of transgene and gene expression were observed in liver, spleen, and bone marrow in all animals. By 40 days after injection, GFP levels had decreased in liver and spleen, but bone marrow exhibited a consistently high level of transgene. This finding was consistent with the increase in both GFP frequency and expression levels observed in peripheral blood by fluorescence-activated cell-sorting (FACS) analysis. Between 0 and 1% transgene was detected in all other organs. No significant pathologic lesions were found attributable to vector in any of the tissues examined. The observation of bone marrow transduction after intravenous vector administration suggests the possibility of an in vivo approach to stem cell gene therapy.

Regression of prostate cancer xenografts by a lentiviral vector specifically expressing diphtheria toxin A

“…Similar results have also been reported by others. 27 Figure 4 Tissue sections of DTA vector-treated and untreated mice. Tail vein injection delivered approximately 0.2 ml of the DTA lentiviral vector with a 3.5 mg p24/ml p24 count.…”

In vivo evaluation of an EIAV vector for the systemic genetic delivery of therapeutic antibodies

“…These observations are in agreement with an earlier study where VSV-G-pseudotyped HIV vectors were found to mainly target the spleen, liver and bone marrow of mice injected i.v. 33,34 Discussion Previous evaluation of HIV-1 lentivectors following systemic delivery have demonstrated long-term expression of the transgene for up to 1 year, depending on the transgene and host. [33][34][35] An alternative to HIV-1 is the EIAV-based vector, which has been developed, in part, because HIV-1 is a human pathogen and as a result may face extra hurdles in clinical use.…”

“…33,34 Discussion Previous evaluation of HIV-1 lentivectors following systemic delivery have demonstrated long-term expression of the transgene for up to 1 year, depending on the transgene and host. [33][34][35] An alternative to HIV-1 is the EIAV-based vector, which has been developed, in part, because HIV-1 is a human pathogen and as a result may face extra hurdles in clinical use. In addition to this, the potential advantages of EIAV are its relative simplicity compared to HIV-1, the inability of the parent virus to replicate in human cells and the nonlethal nature of the infection in its natural host, the genus Equidiae.…”

Gene transfer vector biodistribution: pivotal safety studies in clinical gene therapy development

“…Other techniques include nonquantitative or semiquantitative PCR, FISH (approx. 50%), Southern blot 1,2 and reporter gene expression (LacZ, [1][2][3][4][5][6]7 luciferase, [8][9][10][11] and GFP 12 ).…”

“…Some authors also used an internal control, which is often a competitive piece of DNA used in the same reaction tube. 12,15,[17][18][19][20] The advantage of this technique is a precise control of inhibition in the same tube, the inconvenience being the sensitivity decrease because of competition. We would recommend qPCR in triplicate, with spiking of one of the reaction tubes with a low DNA amount (as in Hanke et al 13 ).…”