Combinatorial RNA-based gene therapy for the treatment of HIV/AIDS

Abstract

Introduction: HIV/AIDS continues to be a worldwide health problem and viral eradication has been an elusive goal. HIV+ patients are currently treated with combination antiretroviral therapy (cART) which is not curative. For many patients, cART is inaccessible, intolerable or unaffordable. Therefore, a new class of therapeutics for HIV is required to overcome these limitations. Cell and gene therapy for HIV has been proposed as a way to provide a functional cure for HIV in the form of a virus/infection resistant immune system.

Areas covered: In this review, the authors describe the standard therapy for HIV/AIDS, its limitations, current areas of investigation and the potential of hematopoietic stem cells modified with anti-HIV RNAs as a means to affect a functional cure for HIV.

Expert opinion: Cell and gene therapy for HIV/AIDS is a promising alternative to antiviral drug therapy and may provide a functional cure. In order to show clinical benefit, multiple mechanisms of inhibition of HIV entry and lifecycle are likely to be required. Among the most promising antiviral strategies is the use of transgenic RNA molecules that provide protection from HIV infection. When these molecules are delivered as gene-modified hematopoietic stem and progenitor cells, long-term repopulation of the patient's immune system with gene-modified progeny has been observed.

Figure 1. Comparison of the HIV genome and pHIV7-based DNA construct and integrated self-inactivating lentiviral vector

We used pHIV7, an HIV-based DNA construct to package lentiviruses as a deliver vehicle for anti-HIV small RNAs. pHIV7 has a hybrid 5’ LTR with the U3 region replaced with the cytomegalovirus (CMV) promoter and enhancer sequence to eliminate the need for HIV-1 Tat protein for transcription during packaging. pHIV7 has majority of the viral genes removed and therefore requires the co-transfection of multiple helper plasmids during packaging. The co-packaging plasmids encoding for HIV-1 Gag/Pol and Rev provide the structural genes required to produce viral particles, while Rev interacts with RRE to reduce intron splicing during packaging and facilitates the export of intron-containing transcripts. The use of Vesicular Stomatitis virus glycoprotein (VSVG) as the envelop expands the tropism of the vectors in addition to allowing concentration via ultracentrifugation. The packaging signal (ψ) is important for encapsidation while the Rev-responsive element (RRE) is required for export of intron-containing transcripts out of the nucleus. The flap sequence is required for nuclear import of the vector cDNA after reverse transcription and is required for transducing non-dividing cells [15]. The woodchuck post-transcriptional regulatory element (WPRE) increases the expression of the transgene by promoting RNA export and/or polyadenylation [16]. Upon integration into the target cells, the 200-bp deletion in the U3 region in the 3’ LTR, denoted ΔU3, is further copied to the 5’ LTR after reverse transcription generating a self-inactivating vector to increase biosafety. The small RNA genes are cloned in the multiple cloning site (MCS) of the vector with the Sh1 targeting tat/rev common exon driven by the U6 promoter, the nucleolar U16TAR decoy driven by the U6 promoter, and finally the CCR5 ribozyme driven by the VA1 promoter.