Generation of More Efficient Lentiviral Constructs with Modified U1snRNA for Anti-HIV-1 Gene Therapy

Matthew Douglas-Vail, Maria Malm, Alan Cochrane, Juan Carlos Zuniga-Pflucker

Abstract


The difficulty in treating drug-resistant strains of HIV-1 has highlighted the need for the development of novel inhibitory methods to complement highly active antiretroviral therapy (HAART). To address this issue, lentiviral vectors were used to transduce human CD34+CD38-/lo hematopoietic stem cells (HSCs) with modified U1snRNAs that suppress HIV-1 RNA processing. Engraftment of transduced HSCs has the potential to generate HIV-1 resistance, as cells normally targeted by HIV-1, CD4+ T cells, dendritic cells and macrophages, are reconstituted by the modified HSCs[1]. These modified U1snRNA constructs contain a 5’ 10-nucleotide substitution, which binds to highly conserved 3’ terminal exon regions of HIV-1. This binding prevents 3’ end formation of pre-mRNA, reducing HIV-1 protein expression and viral replication[2]. However, a limiting factor in this method of gene therapy is the difficulty in obtaining a suitable number of transduced CD34+CD38-/lo HSCs for transplantation. Here we show that anti-HIV-1 snRNA lentiviral vectors can be produced with sufficient titer to transduce up to 37% of cord blood-derived CD34- cells. Three protocols were used to produce lentiviral particles (vectors) carrying different U1snRNA constructs, as well as a GFP reporter gene. The titers of progeny viral vectors were compared in 293T (human embryonic kidney) cells and in cord blood-derived CD34- cells. Dr. Alan Cochrane kindly provided the transfer plasmids, encoding the constructs to be package within the lentiviral particles. The perparation of vector particles produced using the serum-free expression media/ultrafiltration protocol transduced 36.9% of cord-blood derived CD34- cells, compared to viruses from the original protocol, which transduced 7.61% of the cells.

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