Using Lentiviral Vectors as Delivery Vehicles for Gene Therapy

Abstract

Viral-vector-based gene therapy is a powerful tool that allows experimental studies of species that previously were not amenable to genetic manipulation. Nonhuman primates (NHPs) are an invaluable resource for the study of genetic regulation of disease mechanisms. The main disadvantage of using NHPs as a preclinical model of human disease is the diffi culty of manipulating the monkey genome using conventional gene modifying strategies. Lentiviruses offer the possibility of circumventing this diffi culty by infecting and transducing dividing or nondividing cells, without eliciting an immune response. In addition, lentiviruses can permanently integrate into the genome of host cells and are able to maintain long-term expression. In this chapter, we describe the lentiviral vectors that we use to both express transgenes and suppress expression of endogenous genes via RNA interference (RNAi) in NHPs. We also discuss the safety features of currently available vectors that are especially important when lentiviral vectors are used in a species as closely related to humans as NHPs. In addition, we describe in detail the lentiviral vector production protocol we use and provide specifi c examples of how this vector can be employed to target peripheral tissues and the brain. Finally, we conclude by comparing and contrasting the use of lentiviral vectors with adeno-associated viral vectors with a particular emphasis on the use of these vectors in preclinical and clinical studies.

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression

“…These primers encompass the juncture between the GATAD1 cDNA and LV-Ief. The LV-IEF plasmid derives from the plasmid pLVI IRES 49 50 in which the IRES element connecting the gene of interest to eGFP was replaced with the promoter of human elongation factor to drive eGFP expression independent of the inserted gene. pLVI-IRES is a third generation plasmid, in which the promoter sequences of the 5′-LTR were replaced by the cytomegalovirus (CMV) promoter resulting in the formation of a heterologous U3 promoter 51 52 .…”

CRISPR-Cas9: A Promising Genome Editing Therapeutic Tool for Alzheimer’s Disease—A Narrative Review

“…Compared to AAV, lentivirus is comparatively difficult to purify in large quantities and is more likely to provoke immune reactions and integrate into the human genome at high efficiency [ 46 ]. However, incorporation of long DNA inserts (8–10 kb) is possible into lentivirus, but with a lower brain spreading efficiency [ 48 ]. Researchers showed the possibility of using lentivirus to target three different genes in SAD and familial AD, which are APP , APOE E4 , and caspase-6 [ 49 – 51 ].…”

“…Aside from CRISP-Cas9 delivery, other vehicles conveying short interfering RNAs (siRNA) have been created to target AD across the BBB. Polymeric nanocomplexes of poly(mannitol- co -polyethylenimine) carrier (PMT) modified with rabies infection glycoprotein (RVG) have recently been reported [ 48 ]. The polymer is complexed to siRNA against BACE1 .…”

Applying Gene Silencing Technology to Contraception

“…In spite of existing as an episome, the AAV genome can drive transgene expression for long periods of time. Transgenes from AAV vectors can be detected in many different tissues of rodents throughout the lifespan of the animal (Kaplitt et al 1994;McCown et al 1996;Xiao et al 1996); in non-human primates, they have been detected for as long as 10 years (Dissen et al 2012b). Another factor that has contributed to the acceptance of the AAV vector for gene therapy is that the vector is derived from a nonpathogenic virus (Mueller and Flotte 2008;Mingozzi and High 2011).…”