Stable Gene Transfer to Muscle Using Non-integrating Lentiviral Vectors

Abstract

Human immunodeficiency virus (HIV)-based lentiviral vectors (LVs) hold immense promise for gene delivery applications because of their relatively large packaging capacity and their ability to infect a range of cell types. The genome of HIV non-specifically integrates into the host genome, and this promotes efficient, stable transgene expression in dividing cells. However, integration can also be problematic because of variations in gene expression among cells, possible gene silencing and, most importantly, insertional mutagenesis which can lead to undesirable effects such as malignant transformation. In order to alleviate these problems, we have developed a range of non-integrating LVs (NILVs) by introducing point mutations into the catalytic site, chromosome binding site, and viral DNA binding site of the viral integrase (IN). In addition, we have mutated the IN attachment (att) sites within the HIV long terminal repeats (LTRs). All of the vectors produced show efficient reverse transcription and transgene expression in dividing cells and prolonged expression in non-dividing myotubes. Finally, we show that NILV can be used for achieving highly effective gene transfer and expression in muscle in vivo.

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

“…Therefore, we produced identical integrase proficient (IP) and integrase defective (ID) LV and GV SIN vectors expressing eGFP from an internal spleen focus-forming virus (SFFV) promoter, and transduced human (HT1080) and murine (SC-1) fibroblast cells. The usage of ID retroviral vectors allows for transient gene expression 29,30 and may reveal whether a potential restriction targets the integration step.…”

Integrase-defective lentiviral vectors: progress and applications

“…An alteration in any of these residues inactivates the catalytic properties of IN. Of these potential mutation target sites, D64 is most commonly altered, 13,22,44,[48][49][50][51][52][53][55][56][57]60 but mutations to D116 have also been reported. 51,54,58,59 Additional mutations affect IN DNA binding, linear episome processing or IN multimerization ( Figure 2).…”

“…[47][48][49] Several reports have detailed the production of HIV IDLVs. 13,22,44,[47][48][49][50][51][52][53][54][55][56][57][58][59][60] In addition, feline immunodeficiency virus (FIV) IDLVs are also described. 48 Integrase-defective LVs: progress and applications MB Banasik and PB McCray Jr studies ( Figure 1).…”

“…Vector DNA binding is impaired by altering Q148. 50 By mutating the RRK(262-264) motif in the C-terminal domain, IN fails to bind target genomic DNA. 47,50,73 Both N120 and W235 are also implicated in genomic DNA binding and generate IDLV mutations when altered.…”

Transgenic Expression of Human Glial Cell Line-Derived Neurotrophic Factor from Integration-Deficient Lentiviral Vectors is Neuroprotective in a Rodent Model of Parkinson's Disease

“…Hence, IDLVs are ideally suited for applications in the postmitotic CNS environment (Peluffo et al, 2013), but to our knowledge they have not been previously applied in the study of a major disease. Additionally, most experiments using IDLVs have focused on a relatively short time scale (weeks), with very few studies so far confirming efficient and long-lasting CNS gene expression in vivo (Philippe et al, 2006;Yáñez-Muñoz et al, 2006;Apolonia et al, 2007;Rahim et al, 2009;Hutson et al, 2012).…”