Search

Human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and feline immunodeficiency virus (FIV) are capable of packaging viral RNA derived from heterologous as well as homologous lentiviruses, a phenomenon referred to as "cross packaging." To remove the possibility of seroconversion to HIV proteins, and to avoid potential problems arising due to targeting of vector or packaging construct by antiviral genes, we investigated the feasibility of using an FIV-based packaging system to deliver human immunodeficiency virus type 2 (HIV-2)-based vectors bearing anti-HIV-1 RNA expression cassettes to target cells. In the absence of FIV rev, FIV was packaged by HIV-2 at only 3% the efficiency of FIV packaging by FIV, but this was increased to 39% of homologous controls by supplying FIV rev in trans. HIV-2 vectors were packaged by FIV at levels equal to or exceeding the homologous HIV-2 packaging system in the absence of HIV-1 tat and rev, and levels increased approximately four- to fivefold with the addition of tat and rev in trans. HIV-2 vectors bearing a polyribozyme cassette targeting multiple regions of HIV RNA were efficiently packaged by FIV and transferred to target cells. Upon challenge with cell-free HIV-1 (m.o.i. = 0.1) a significant reduction in replication was observed. These findings demonstrate that packaging HIV vectors with FIV is a viable alternative, which avoids use of HIV structural proteins.

Minimal vectors derived from the feline immunodeficiency virus (FIV) were constructed which retained only 259 bp of gag cds, the FIV RRE, and incorporated the woodchuck posttranscriptional regulatory element (wPRE), human immunodeficiency virus (HIV) or FIV cPPT/CTS elements, and a variety of eGFP reporter gene promoters (including CMV IE, EF1a, PGK, MSCV, and MPSV). A packaging plasmid (pC34N) deleted of 1569 bp in the env gene and the principal packaging site was used to generate vectors pseudotyped with the VSV-G protein after transfection into 293FT cells. These second generation FIV vectors generated high titers of 106 TU/ml (up to 109 TU/ml upon concentration), and transduced primary human cord blood CD34+ cells at efficiencies of 50–75% (moi 20-40). Expression persisted at the level of 15–20% after 12 days in culture. Vectors containing the EF1a promoter and cPPT /CTS elements transduced CD34+ cells most efficiently. While the FIV LTR is relatively quiescent in human cells (no LTR-driven expression detected in transduced cells expression by RT PCR), "self-inactivating" 3rd generation FIV vectors were constructed by progressive deletion of the U3 in the 3'LTR, encompassing TATA boxes and binding sites for NF-kB, AP1 and C/EBP, in an attempt to increase expression. One FIV SIN vector had 2–3 fold increase in titer, higher mean fluorescence intensity and improved transduction efficiency of CD34+ cells. FIV vector transduced CD34+ cells expressed eGFP upon differentiation into myeloid cells. FIV SIN transduced CD34+ cells gave rise to GFP+ colonies which persisted for more than 3 weeks when plated onto methylcellulose for hematopoietic progenitor cell assays. Integration assay (two -step Alu PCR) demonstrated that these vectors were integrated into the genome. FIV SIN vectors have potential as an alternative to HIV-1 based vectors for stem cell gene therapy.

Top of pageAbstract Silencing of expression produced by small interfering RNA (siRNA) directed against different regions of a lentiviral vector transgene expressing enhanced green fluorescent protein (EGFP) driven by the elongation factor 1 alpha (EF1a) promoter 12 was investigated in transfected and transduced 293FT fibroblasts and human peripheral mononuclear cells. SiRNA directed against exonic sequences significantly reduced expression, including one targeting exon 1 (+10 to +30 relative to the transcriptional start site), and one targeting EGFP coding sequences (+1167 to +1185). Unexpectedly, siRNA directed against intron A (+155 to +175 relative to the transcriptional start site) or targeting the EF1a promoter (−106 to −86 and −25 to −5 start) also silenced EGFP expression. Post-transcriptional gene silencing in human cells has been described to be restricted to the cytoplasm 3, and transcriptional gene silencing (TGS) mediated by RNA interference (RNAi) has not previously been shown to occur in mammalian cells. Silencing of expression could not be attributed to reduced vector transduction, non-specific inhibition of transcription, cytotoxicity, initiation from a cryptic promoter, or aberrant splicing of EGFP mRNA, and was confirmed to occur at the level of transcription by nuclear run-on. Endogenous EF1a expression could inhibited by transfecting siRNA using a reagent (MPG)4w which facilitates nuclear entry. Inhibition of expression and methylation of one site in the promoter was reversible with addition of 5′ azacytidine and trichostatin A. Treatment with siRNA targeting intronic sequences and promoter regions can induce transcriptional gene silencing. Access of siRNA to the nucleus may be key to the observed effects.