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. 2009;44(1-3):112-26.
doi: 10.1007/s12026-008-8088-z.

Suppression of HLA expression by lentivirus-mediated gene transfer of siRNA cassettes and in vivo chemoselection to enhance hematopoietic stem cell transplantation

Katrin Hacke  1 , Rustom Falahati, Linda Flebbe-Rehwaldt, Noriyuki Kasahara, Karin M L Gaensler
Affiliations

Suppression of HLA expression by lentivirus-mediated gene transfer of siRNA cassettes and in vivo chemoselection to enhance hematopoietic stem cell transplantation

Katrin Hacke et al. Immunol Res. 2009.

Abstract

Current approaches for hematopoietic stem cell (HSC) and organ transplantation are limited by donor and host-mediated immune responses to allo-antigens. Application of these therapies is limited by the toxicity of preparative and post-transplant immunosuppressive regimens and a shortage of appropriate HLA-matched donors. We have been exploring two complementary approaches for genetically modifying donor cells that achieve long-term suppression of cellular proteins that elicit host immune responses to mismatched donor antigens, and provide a selective advantage to genetically engineered donor cells after transplantation. The first approach is based on recent advances that make feasible targeted down-regulation of HLA expression. Suppression of HLA expression could help to overcome limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors. Accordingly, we have recently investigated whether knockdown of HLA by RNA interference (RNAi) enables allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors that integrate into genomic DNA, thereby permanently modifying transduced donor cells. Lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA achieved efficient and dose-dependent reduction in surface expression of HLA in human cells, and enhanced resistance to allo-reactive T lymphocyte-mediated cytotoxicity, while avoiding non-MHC restricted killing. Complementary strategies for genetic engineering of HSC that would provide a selective advantage for transplanted donor cells and enable successful engraftment with less toxic preparative and immunosuppressive regimens would increase the numbers of individuals to whom HLA suppression therapy could be offered. Our second strategy is to provide a mechanism for in vivo selection of genetically modified HSC and other donor cells. We have uniquely combined transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of mechanisms of tolerance induction to neo-antigens, and allogeneic stem cells during immune ontogeny. HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O(6)-methylguanine-methyltransferase (MGMT(P140K)). The MGMT(P140K) DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. In vivo chemoselection enables enrichment of donor cells at the stem cell level. Using complementary approaches of in vivo chemoselection and RNAi-induced silencing of HLA expression may enable the generation of histocompatibility-enhanced, and eventually, perhaps "universally" compatible cellular grafts.

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Figures

Figure 1
Figure 1. Targeting HLA Class I to reduce graft immunogenicity
Three potential mechanisms for allograft rejection of graft cell A, resulting in its elimination are depicted: 1) direct antigen recognition, in which T cell receptors (TCR) on host T cells recognize intact donor HLA molecules on the graft cells as non-self, presumably because their three-dimensional structure resembles a self MHC bound to a foreign peptide (“molecular mimicry”), 2) peptides derived from donor HLA molecules are presented by host antigen-presenting cells (APC) as foreign antigens, 3) host antibodies against donor HLA bind and initiate graft damage through antibody-dependent cellular toxicity and complement activation. Graft cell B depicts the proposed scenario if HLA expression is silenced: although co-stimulatory molecules such as CD80 (2nd signal) might be displayed, none of the above mechanisms would be activated in the absence of HLA on the donor-derived graft cells. This could lead to prolonged survival by making the graft cells invisible to allo-reactive immune responses, but could also incur MHC-non-restricted killing by host NK or LAK cells.
Figure 2
Figure 2. Molecular events associated with retrovirus or lentivirus vector -mediated transduction
(asterisks denote aspects specific to lentiviral vectors). 1: Virion adsorption via interaction between viral envelope protein and cell surface receptor (* lentiviral vectors, and in some cases retroviral vectors, can be pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) envelope, allowing broad tropism by binding directly to phospholipids). 2: Virus-cell lipid membrane fusion, allowing entry of viral nucleocapsid complex into cytoplasm. 3: Reverse transcription of viral genomic RNA (single line) to double-stranded DNA (double lines), U3 and U5 sequences duplicated at 5′ and 3′ ends, respectively, to convert R-U5 and U3-R into matching long terminal repeat (LTR) sequences flanking viral genome. 4: Entry into cell nucleus, either by passive diffusion upon nuclear membrane breakdown during mitosis (oncoretrovirus) or *active uptake by recognition of nuclear localization signal (NLS; lentivirus). 5: Permanent integration of proviral DNA into host cell chromosome, resulting in stable long-term transduction.
Figure 3
Figure 3. Lentiviral vector constructs for short hairpin RNAs directed against HLA
The basic lentiviral vector pLentiLox-DsRed construct contains a U6 promoter that can drive expression of a short hairpin siRNA (shRNA; sense siRNA-loop-antisense siRNA) sequence, and a downstream CMV promoter-driven red fluorescent marker gene (DsRed) cassette. LTR: lentiviral long terminal repeat, ψ: packaging signal. Shown below are the allele-specific siRNA sequences (designed against HLA- A2.1 (A*020101 allele)) and pan-specific siRNA sequences (against conserved regions in HLA-A, B, C) that were designed and tested in the pLentiLox vector. Arrows indicate the sequences showing the best knockdown activity that were selected for further testing.
Figure 4
Figure 4. In vitro gene delivery of MGMT into HSC followed by transplantation and in vivo chemoselection
Donor HSC are isolated (Step 1) and transduced in vitro with lentivirus containing the MGMTP140K –GFP transgene (MAG vector) resulting in integration into chromosomal DNA (Step 2). Subsequently, neonatal mice are preconditioned with a non-ablative regimen (Step 3) and MAG transduced HSC are delivered via intravenous injection (Step 4). Following engraftment, in vivo chemoselection is performed by delivery of BG followed by BCNU (Step 5). Initially the graft consists of a small numbers of MAG-transduced HSC. Sequential chemoselection (Step 6) results in apoptosis of untransduced HSCs and enrichment of MAG-transduced HSC. Enriched MAG- transduced HSC expand and repopulate all hematopoietic lineages.
Figure 5
Figure 5. In vitro and In vivo chemoselection of syngenic and allogeneic HSC
(A) Lentivirus MAG transduced (> 90% expressing MGMTP140K) or untransduced 293 T cells were incubated with 10 uM BG and 50 uM BCNU for 6 days. Viability of cells on Day 6 following treatment was evaluated by using the MTS assay (Promega). Percent survival is shown for each group as compared to untransduced control cells without drug treatment. (B) BALB/c whole BM cells (5 × 105) were transduced overnight after prestimulation and transplanted into BALB/c neonates preconditioned with a non-myleoablative regimen. BG (30mg/kg)/ BCNU (7.5mg/kg) was administered 5 and 11 weeks post transplant. Flow analysis of peripheral blood was performed immediately prior to the initiation of chemoselection (light gray), and at 11 (dark gray), and 15 weeks post transplant (Black curve). (C) BALB/c whole BM cells (1.7 × 106) were transduced by spinoculation and transplanted into 2 day-old C57BL/6 X BALB/c F1 neonates preconditioned with a non-myleoablative regimen. BG (30mg/kg)/BCNU (7.5mg/kg) was administered 5 and 10 weeks post transplant. Flow analysis of peripheral blood was performed immediately prior to the initiation of chemoselection (light gray curve), and at 10 weeks (dark gray curve), and 15 weeks (black curve) post transplant.
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