Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases

Abstract

Lentiviral vectors are the most frequently used tool to stably transfer and express genes in the context of gene therapy for monogenic diseases. The vast majority of clinical applications involves an ex vivo modality whereby lentiviral vectors are used to transduce autologous somatic cells, obtained from patients and re-delivered to patients after transduction. Examples are hematopoietic stem cells used in gene therapy for hematological or neurometabolic diseases or T cells for immunotherapy of cancer. We review the design and use of lentiviral vectors in gene therapy of monogenic diseases, with a focus on controlling gene expression by transcriptional or post-transcriptional mechanisms in the context of vectors that have already entered a clinical development phase.

Keywords: ex vivo gene therapy; lentiviral vectors; miRNA; post-transcriptional regulation; promoters; retroviral integration; transcriptional regulation.

Figure 1

Schematic representation of HIV-1-derived lentiviral vectors (LVs) used in clinical applications of gene therapy for monogenic diseases. (A) A prototype, third-generation LV provirus, featuring a disabling (SIN) deletion of the enhancer/promoter sequences in the U3 region of the long terminal repeat (LTR) up to position -18 from the viral transcription start site. The enlarged portion of the LTR shows the original arrangement of the transcription factor binding sites and TATA box in the HIV-1 U3 region, deleted in the LV LTR. The poly(A) signal-containing R and the U5 regions of the LTR are retained. PBS, primer binding site; ΔGAG, deleted, non-coding portion of the GAG gene containing the D1 major HIV-1 splice donor (SD) site (CTG/GTGAGTAC); RRE, Rev-responsive element; ΔENV, deleted portion of the ENV gene containing the A7 HIV-1 splice acceptor (SA) site (TCGTTTCAG/A); cPPT, central polypurine tract; E/P, enhancer/promoter component of the expression cassette (the arrow represents the transcription start site); WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; Ψ, extended packaging signal. (B) 1 to 10, schematic composition of the LVs described in the text. MND, modified enhancer/promoter of the murine myeloproliferative sarcoma virus; ABCD1, ATP-binding cassette, subfamily D, member 1 cDNA; EF1α, short promoter of the elongation factor 1α gene; ADA, Adenosine deaminase cDNA; W, WPRE; PGK, phosphoglycero kinase gene promoter; ARSA, arylsulfatase cDNA; WASPp, 1.6-kb extended Wiskott-Aldrich protein gene promoter; WASP, Wiskott-Aldrich protein cDNA; CTSG-cFES, hybrid promoter containing cFES regulatory regions and CTSG promoter. GP91, Gp91^phox cDNA; MSP, myeloid-specific promoter; 126T (2×) tandem repeat of the miR-126 binding site; ET, liver-specific transthyretin promoter/enhancer; FIX, coagulation factor IX cDNA; 142T (4×) tetrameric repeat of the miR-142 binding site; E3, E2, E1, exons 3, 2 and 1 of the human β-globin gene; βp, human β-globin gene promoter; HS2, HS3, HS4, hypersensitive site 2, 3 and 4 regions of the β-globin locus-control region; 3′ Enh, 3′ enhancer of the human β-globin gene. T87Q, codon substitution causing a tryptophan to glutamine amino acid substitution at position 87 of the β-globin protein; miRNA, gene encoding a shRNA/miR hybrid small nuclear RNA targeting the BCL11A transcription factor mRNA.