Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery

Abstract

In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3′ long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.

Retroviral Vectors Pseudotyped with Severe Acute Respiratory Syndrome Coronavirus S Protein

“…Lentiviral vectors were produced likewise by transfecting 293T cells with 7.5 g of packaging plasmid pCMV⌬R8.91 (encoding human immunodeficiency virus [HIV] Gag/Pol), 12.5 g of eGFP harboring transfer vector pHRЈSIN.cPPT-SEW (7), and 1 to 4 g of env plasmids. The lentiviral transfer vector is a self-inactivating HIV type 1 (HIV-1)-derived vector (46) which contains the posttranscriptional regulatory element of woodchuck hepatitis virus to enhance transgene expression as well as a central polypurine tract to improve gene transfer efficiency. The expression of eGFP is driven by the spleen focus-forming virus promoter.…”

The Novel Replication-defective Vaccinia Virus (Tiantan Strain)–based Hepatitis C Virus Vaccine Induces Robust Immunity in Macaques

“…21,33,34 The gene was conventionally subcloned into the poxvirus shuttle vector pJSA1175 derived in our laboratory, which places the insert gene under the control of the vaccinia virus p7.5 promoter and provides flanks for recombination at the TK locus of NTV (Figure 1a). 21,[35][36][37][38] NTVJTK + ΔCK, replication-defective Tiantan strain vaccinia virus(NTV) with the CK region deleted (Supplementary Figure S1), was used as the original virus. 15,39 Recombinant viruses were isolated following transfection of this plasmid into infected primary chick embryo fibroblasts (day 9 of gestation) by transient dominant selection using LacZ.…”

“…40 E1E2(161-746 aa) or NS3 was inserted into the pVRC envelope plasmid (pVRC-E1E2) and transducing plasmid pCS (pCS-NS3). 38 The integration-deficient packaging plasmid pCMVΔR8.2D64E, which contained a point mutation in the integrase (D64E) domain of human immunodeficiency virus (HIV), [41][42][43] was constructed in our laboratory. Three plasmids (pCMVΔR8.2D64E, pCS-NS3, pVRC-E1E2) were cotransfected into human embryonic kidney (HEK) 293FT cells using a Fugen HD reagent (Roche, Basel, Switzerland).…”

“…The HCVpp (Figure 1c) was harvested 48 hours after transfection, and the virus titre was presented as the HIV p24 antigen concentration determined using the Vironostika HIV-1 Antigen MicroELISA System (BioMérieux, Shanghai, China). 38 Animals and immunizations. Rhesus macaques were housed at the Laboratory Animal Centre in the Academy of the Military Medical Sciences of the PLA (AMMS).…”

Generation of MERS-CoV Pseudotyped Viral Particles for the Evaluation of Neutralizing Antibodies in Mammalian Sera

“…They have been used to develop pseudotype viral particles for many pathogenic viruses [3][4][5][6]. These replication-deficient vectors offer a number of advantages including that they do not replicate in mammalian cells, they infect dividing and nondividing cells, they can incorporate large transgenes derived from other pathogenic viruses as large as 9 kb, and they induce no or weak immune response [7][8][9]. Several studies have utilized lentiviral vectors to generate MERS-CoV pseudotype viral particles (MERSpp) to evaluate nAbs in humans and animals.…”