Garry P. Larson

INHIBITION OF HIV-1 IN HUMAN T-LYMPHOCYTES BY RETROVIRALLY TRANSDUCED ANTI-TAT AND REV HAMMERHEAD RIBOZYMES

Gene therapy for AIDS requires the identification of genes which effectively inhibit HIV-1 replication coupled to an efficient vector system for gene delivery and expression. Hammerhead ribozymes are RNA molecules capable of catalytic cleavage of complementary RNA molecules. Ribozymes targeted against two portions of the HIV-1 genome were designed to cleave HIV RNA in the tat gene (TAT) or in a common exon for tat and rev (TR). The ribozymes were cloned into the LN (LTR-neomycin) retroviral vector plasmids and expressed as part of viral LTR-driven transcripts. The vectors were packaged as amphitropic virions and used to transduce human T-lymphocytes. Expression of the vector transcripts containing the ribozyme sequences was readily detected by Northern blot analysis of the transduced T cells. The T-lymphocytes expressing the anti-HIV-1 ribozymes showed resistance to HIV-1 replication. In contrast, cells expressing mutant ribozymes, containing substitutions of a key nucleotide in the catalytic domain which cripples the cleavage activity of the ribozymes, supported replication of HIV-1, demonstrating that the functional ribozymes were cleaving the target RNAs. These studies demonstrate that retrovirally transduced ribozymes included in long, multifunctional transcripts, can inhibit HIV replication in human T-lymphocytes. The ribozyme and expression strategies described here should be useful for the gene therapy of AIDS by conferring resistance to HIV-1 replication on cells derived from transduced hematopoietic stem cells.

Identification of disease gene variants based on gene–gene interactions

New methods are needed for the identification of pathogenic alleles of candidate genes that may increase cancer susceptibility. Such risk alleles are expected to be of low penetrance and may act alone or modify the effects of other genes. We have developed a method that enriches for pathogenetic disease variants contingent on gene−gene interactions. Candidate gene pairs are chosen based on previous evidence demonstrating genetic or biochemical interaction. Utilizing a cohort of sibling pairs affected with breast cancer, we tested this paradigm by identifying risk variants of cell cycle control gene CDKN1A by means of interactions with TP53 and BRCA1. This approach, which we call disease association by locus stratification, first stratified affected pairs based on sharing of both alleles at a microsatellite marker linked to CDKN1A. The second stratification was based on microsatellite marker sharing at TP53 or BRCA1. We identified subsets of affected pairs sharing both alleles at both loci as screening targets. Utilizing this approach, we were able to enrich for two noncoding disease haplotypes of CDKN1A by virtue of BRCA1 interactions. We defined each haplotype by single-nucleotide polymorphisms at two positions potentially important in CDKN1A transcriptional activation by both p53 and BRCA1p. Our results indicated that an approach based on allele sharing and gene−gene interactions will be valuable not only in identifying risk alleles but also in elucidating their mechanism of action.