Optimized lentiviral vectors for HIV gene therapy: multiplexed expression of small RNAs and inclusion of MGMT(P140K) drug resistance gene

Abstract

Gene therapy with hematopoietic stem and progenitor cells is a promising approach to engineering immunity to human immunodeficiency virus (HIV) that may lead to a functional cure for acquired immunodeficiency syndrome (AIDS). In support of this approach, we created lentiviral vectors with an engineered polycistronic platform derived from the endogenous MCM7 gene to express a diverse set of small antiviral RNAs and a drug resistance MGMT(P140K) marker. Multiple strategies for simultaneous expression of up to five RNA transgenes were tested. The placement and orientation of each transgene and its promoter were important determinants for optimal gene expression. Antiviral RNA expression from the MCM7 platform with a U1 promoter was sufficient to provide protection from R5-tropic HIV in macrophages and resulted in reduced hematopoietic toxicity compared with constructs expressing RNA from independent RNA polymerase III promoters. The addition of an HIV entry inhibitor and nucleolar TAR RNA decoy did not enhance antiviral potency over constructs that targeted only viral RNA transcripts. We also demonstrated selective enrichment of gene-modified cells in vivo using a humanized mouse model. The use of these less toxic, potent anti-HIV vectors expressing a drug selection marker is likely to enhance the in vivo efficacy of our stem cell gene therapy approach in treating HIV/AIDS.

Figure 1

Strategy overview on multiplexing small RNAs with different classes of promoters. (a) pHIV7 lentiviral vector with MGMT^P140K selectable marker (pLV). We utilized a third-generation HIV-1–based lentiviral vector (pHIV7) with enhanced green fluorescent protein (EGFP) marker to label gene-modified cells. The chemical resistance MGMT^P140K marker is coexpressed with EGFP with a self-cleaving P2A peptide with the cytomegalovirus (CMV) promoter. (b) First-generation lentiviral vector (FGLV) with small RNAs expressed from independent RNA polymerase III (Pol III) promoters. (c) Second-generation lentiviral vector (SGLV) with small RNAs expressed in the MCM7 polycistronic platform with single RNA polymerase II (Pol II) promoter. The naturally occurring microRNA cluster in the intron of the human MCM7 gene was engineered to coexpress different classes of antiviral small RNAs with single Pol II U1 promoter for ubiquitous transgene expression in all hematopoietic cells. Independent Pol III driven RNA cassettes can be incorporated for expression of up to five small RNAs. The tRNA^Ser-CCR5sh cassette was incorporated into the 3′ intron of MCM7 in both orientations (“F” Forward and “R” Reverse) with respect to the parental U1 promoter. The U6-U16TAR cassette was cloned downstream of the U1 termination signal (U1t). Promoters: CMV, cytomegalovirus promoter (Pol II); U6, human small nuclear U6 promoter (Pol III); VA1, adenoviral promoter (Pol III); U1, human small nuclear U1 promoter (Pol II); tRNA^Ser, human transfer RNA Serine promoter (Pol III). Small RNA transgenes: S1, tat/rev siRNA; S2M, rev siRNA; S3B tat, siRNA; CCR5sh, CCR5-targeting short hairpin RNA; U16TAR, nucleolar TAR RNA decoy; U16U5RZ, nucleolar U5-targeting ribozyme; CCR5RZ, CCR5-targeting ribozyme. HIV, human immunodeficiency virus.

Figure 2

Biological activity of tRNA^Ser-CCR5sh cassette. (a) Potent CCR5 knockdown in U373-MAGI-CCR5E cells. Plasmid with only the tRNA^Ser promoter sequence (solid black line) or with the tRNA^Ser-CCR5sh cassette (dashed gray line) was transiently transfected into CCR5 over-expressing U373-MAGI-CCR5E cells with knockdown estimated by flow cytometry 72 hours later. Potent and specific downregulation of surface CCR5 expression was only observed with the construct containing CCR5sh. (b) Potent CCR5 RNA transcript degradation in gene-modified macrophages. Adult hematopoietic stem and progenitor cells were transduced with the indicated lentiviral vectors, sorted based on enhanced green fluorescent protein expression, and then differentiated into macrophages as described in Material and Methods. CCR5 transcript knockdown was measured by quantitative reverse transcription polymerase chain reaction with normalization to GAPDH housekeeping gene and expressed relative to the untransduced control. tRNA^Ser-CCR5sh cassette in the context of MCM7 platform induced potent silencing in gene-modified macrophages. tRNA^Ser, transfer RNA Serine promoter.

Figure 3

Optimization of the tRNA^Ser-CCR5sh cassette expression in the MCM7 platform. (a) Orientation dependence of the tRNA^Ser-CCR5sh cassette in MCM7. The tRNA^Ser-CCR5sh cassette was cloned in 3′ intron of MCM7 in either the forward (SGLV3) or reverse (SGLV4) orientation then transiently transfected into human embryonic kidney (HEK) 293 cells to evaluate transgene expression and processing by northern blotting. Northern blotting distinguishes products in various steps of processing due to difference in size (tRNA^Ser-CCR5sh fusion transcript (~130–140 nt) that requires tRNase Z processing; short hairpin RNA (shRNA; ~50–60 nt) that requires Dicer processing; small interfering RNA (siRNA; 20–23 nt) represents the completely processed mature siRNA capable of mediating silencing). In this case, the probe detected the guide strand that mediates CCR5 silencing. Northern blotting demonstrated that tRNA^Ser-shRNA cassette is efficiently processed by the RNA interference pathway as the mature siRNA is the predominate product. Furthermore, SGLV4 gives 2.4-fold greater expression in comparison to the opposite orientation after normalization with the loading control U2A RNA. (b) Placement dependence of the tRNA^Ser-CCR5sh cassette. Placement of tRNA^Ser-CCR5sh cassette in the lentiviral vector dramatically affects transgene expression in sorted stably expressing CEM T lymphocytes. Although the CCR5sh cassette is driven independently from the tRNA^Ser promoter, the expression was much lower in the context of MCM7 platform (“inside of MCM7”) compared to as a separate entity (“No MCM7”). In the latter scenario, overexpression is evident by the presence of unprocessed products (i.e., bands representing tRNA^Ser-CCR5sh fusion transcript and shRNA). nt, nucleotide; SGLV, second-generation lentiviral vector; tRNA^Ser, transfer RNA Serine promoter.

Figure 4

Northern blot of stably expressing CEM T lymphocytes demonstrates efficient processing and expression of small RNAs. CEM T lymphocytes were transduced with the indicated lentiviruses then sorted by enhanced green fluorescent protein expression to create stably expressing cell lines. Small RNA transgenes were detected by P³²-labeled probes. U2A small nuclear RNA serves as a loading control. S1, S2M, S3B, and CCR5sh represent fully processed small interfering RNA that are 20-23 nucleotides. U16U5RZ and U16TAR are U16 small nucleolar RNA chimeras that are ~132 nucleotides. CCR5RZ is ~230 nucleotides. Lanes 1: untransduced (unTDX); 2: pLV; 3: SGLV4; 4: SGLV5; 5: SGLV6; 6: FGLV.

Figure 5

Intracellular human immunodeficiency virus type 1 (HIV-1) staining demonstrates potent antiviral protection of macrophages derived from gene-modified hematopoietic stem and progenitor cells (HSPCs). Macrophages differentiated from adult HSPCs transduced with indicated lentiviral vectors were challenged with HIV-1 Bal at a multiplicity of infection of 0.01. Viral infection was monitored by intracellular staining by flow cytometry with an antibody specific to HIV-1 core proteins. Data from 18 days postinfection are shown. Background signal for intracellular staining was established with an uninfected control with identical culture and staining protocol. Intracellular staining showed a high degree of infection in unprotected macrophages, with some constructs with intermediate protection while differentiating some with excellent protection. EGFP, enhanced green fluorescent protein; SGLV, second-generation lentiviral vector. (a) Untransduced and uninfected, (b) untransduced and infected with HIV, (c-i) cells transduced with the indicated lentiviral vector and infected with HIV.

Figure 6

Kinetics of R5-tropic HIV-1 Bal infection in adult hematopoietic stem and progenitor cell (HSPC) derived macrophages monitored by intracellular HIV staining. Kinetics of HIV-1 Bal infection in macrophages differentiated from gene-modified HSPCs were followed by intracellular HIV staining for a total of 42 days to evaluate long-term protection and viral breakthrough. Overexpression of therapeutic small RNAs with independent Pol III promoters (first-generation lentiviral vector (FGLV)) provided potent protection for up to 28 days but eventual breakthrough. In the long term, SGLV2 provided the longest protection with the incorporation of tRNA^Ser-CCR5sh (SGLV4) and U6-U16TAR (SGLV7) cassettes less optimal. D, day; HIV, human immunodeficiency virus; SGLV, second-generation lentiviral vector; tRNA^Ser, transfer RNA Serine promoter.

Figure 7

In vitro colony forming unit (CFU) assay to identify vector toxicity on hematopoietic potential. Transduced hematopoietic stem and progenitor cells were sorted on CD34⁺/EGFP expression after expansion with SR1. A total of 500 sorted cells per sample were plated on methylcellulose medium in triplicate with number of colonies counted 12–13 days later. The absolute number of CFUs was normalized to the respective donor to account for differences in hematopoietic potential in donor viability. Results represent data from at least two independent donors with significant results shown. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. EGFP, enhanced green fluorescent protein.

Figure 8

In vivo drug selection enhances the frequency of gene-modified cells in the bone marrow and spleen of humanized NSG mice expressing MGMT^P140K. Analysis of NSG mice transplanted with gene-modified hematopoietic stem and progenitor cells (HSPCs) expressing MGMT^P140K and treated with two or three doses of O⁶-benzylguanine (O⁶-BG)/bis-chloroethylnitrosourea (BCNU) as described in Material and Methods. Each animal received 1 × 10⁶ HSPCs following transduction with FGLV at the date of transplantation and 20 µg of Fc/IL-7 protein weekly for 11 weeks. (a) Frequency of CD45⁺ cells in the bone marrow of treated mice. (b) Frequency of CD45⁺/EGFP⁺ cells in the bone marrow of treated mice. (c) Frequency of CD45⁺ cells in the spleens of treated mice. (d) Frequency of CD45⁺/EGFP⁺ cells in the spleen of treated mice. (e) Frequency of CD3⁺/CD4⁺/EGFP⁺ gene-modified T lymphocytes in the spleen of treated mice (gated on CD45⁺ population). (f) Frequency of CD14⁺/CD4⁺/EGFP⁺ gene-modified monoctyes in the spleen of treated mice (gated on CD45⁺ population). *P < 0.05, **P < 0.01, ***P < 0.001, ****p<0.0001. EGFP, enhanced green fluorescent protein; IL, interleukin.