Article

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

Department of Medicine, David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, CA, USA.
Immunologic Research (Impact Factor: 3.53). 01/2009; 44(1-3):112-26. DOI: 10.1007/s12026-008-8088-z
Source: PubMed

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.

0 Followers
 · 
99 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Islet transplantation has the potential to cure type 1 diabetes. Despite recent therapeutic success, it is still not common because a large number of transplanted islets get damaged by multiple challenges including instant blood mediated inflammatory reaction, hypoxia/reperfusion injury, inflammatory cytokines, and immune rejection. RNA interference (RNAi) is a novel strategy to selectively degrade target mRNA. The use of RNAi technologies to downregulate the expression of harmful genes has the potential to improve the outcome of islet transplantation. The aim of this review is to gain a thorough understanding of biological obstacles to islet transplantation and discuss how to overcome these barriers using different RNAi technologies. This eventually will help improve islet survival and function post transplantation. Chemically synthesized small interferring RNA (siRNA), vector based short hairpin RNA (shRNA), and their critical design elements (such as sequences, promoters, and backbone) are discussed. The application of combinatorial RNAi in islet transplantation is also discussed. Last but not the least, several delivery strategies for enhanced gene silencing are discussed, including chemical modification of siRNA, complex formation, bioconjugation, and viral vectors.
    Advanced drug delivery reviews 12/2010; 63(1-2):47-68. DOI:10.1016/j.addr.2010.11.003 · 12.71 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The last 40 years has seen the emergence of hematopoietic stem cell transplantation as a therapeutic modality for fatal diseases and as a curative option for individuals born with inherited disorders that carry limited life expectancy and poor quality of life. Despite the rarity of many primary immunodeficiency diseases, these disorders have led the way toward innovative therapies and further provide insights into mechanisms of immunologic reconstitution applicable to all hematopoietic stem cell transplants. This article represents a historical perspective of the early investigators and their contributions. It also reviews the parallel work that oncologists and immunologists have undertaken to treat both primary immunodeficiencies and hematologic malignancies.
    Hematology/oncology clinics of North America 02/2011; 25(1):1-15. DOI:10.1016/j.hoc.2010.11.001 · 2.07 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: RNA interference (RNAi), the process by which double stranded RNA induces the silencing of endogenous genes through the degradation of its correspondent messenger RNA, has been used for post-transcriptional gene silencing allowing scientists to better understand gene function, becoming a powerful tool in reverse genetics for in vivo and in vitro systems. Successful results in vivo have been obtained from invertebrate animal models, whereas vertebrate systems have been limited primarily to mammalian models and cell lines. Nevertheless, exciting results have also been reported from non-mammalian vertebrate models, such as the knock-down of endogenous genes in Xenopus tadpoles by a construct containing both a Xenopus-specific shRNA sequence and the human Ago2 (which is a key enzyme in the RNAi silencing complex), or the design of a novel vector expressing a miRNA driven by a tissue-specific promoter in zebrafish, and the use of an avian retroviral vector to deliver miRNA and shRNA in chicken embryos proving to be effective in knocking-down endogenous genes with a long lasting effect, to mention some examples. Whether dsRNA is able to initiate a specific RNAi response, or all the factors required for RNAi are present in non-mammalian vertebrates, are still questions which remain to be answered. Further progress in understanding natural RNAi mechanisms in non-mammalian vertebrates will help scientists to overcome difficulties and improve this gene silencing technology. There is no doubt that in few years RNAi silencing approaches will become the tool of choice to knock-down genes in all groups of non-mammalian vertebrates, fulfilling different purposes, from basic research to animal therapeutics and drug discovery.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 09/2011; 728(3):158-71. DOI:10.1016/j.mrrev.2011.09.001 · 4.44 Impact Factor
Show more

Preview

Download
0 Downloads
Available from