Inhibition of human immunodeficiency virus-1 entry using vectors expressing a multimeric hammerhead ribozyme targeting the CCR5 mRNA (original) (raw)
Related papers
Gene Therapy, 1997
Retroviral vectors were engineered to express monomeric be detected in these cells and in their culture supernatants and multimeric hammerhead ribozymes targeting one and for up to 60 days after infection. The genomic DNA from nine highly conserved sites within the HIV-1 envelope Rz Env1-9-expressing cells was shown to contain HIV-1 pro-(Env) coding region. In vitro, both the monomeric and multi-viral DNA sequences at days 3 and 60 after HIV infection. meric ribozymes were shown to be active and cleave the HIV-1 used in the challenge experiments was found to contarget RNA containing the cleavage sites. A human CD4 + tain fully reverse transcribed '−' strand DNA which should T lymphocyte-derived MT4 cell line was stably transduced have been able to infect, complete proviral DNA synthesis with retroviral vectors expressing these ribozymes. and integrate within the cellular genome without being Ribozyme expression in stably transduced cells was con-affected by pre-existing ribozymes. Therefore, the proviral firmed by Northern blot analysis and reverse-transcription DNA present at day 3 after infection may have originated polymerase chain reaction (RT-PCR). As compared with from infection by such DNA-containing virus particles. The the control cells lacking any ribozyme, HIV-1 replication results obtained with the retroviral vector expressing was delayed in monomeric Rz Env-expressing cells. Virus Rz Env1-9 are very encouraging and we envisage its future replication was almost completely inhibited in multimeric use in anti-HIV-1 gene therapy. Rz Env1-9-expressing cells as no viral RNA or protein could
Frontiers in Bioscience, 2006
Introduction 3. Materials and Methods 3.1. Construction of retroviral vectors 3.2. Transduction and selection of stable MT4 transductants 3.3. PCR analysis of genomic DNA from MT4 transductants 3.4. RT-PCR analysis of total RNA from MT4 transductants 3.5. In vitro cleavage activity of multimeric ribozymes amplified from the pools of MT4 transductants 3.6. HIV-1 susceptibility of MT4 transductants 4. Results 4.1. MGIN-based vectors expressing the multimeric ribozymes, the antisense RNA, or the multimeric ribozymes and the antisense RNA 4.2. Development of pools of stable MT4 transductants expressing the interfering RNAs 4.3. HIV-1 susceptibility of MT4 transductants expressing the interfering RNAs 5. Discussion 6. Acknowledgements 7. References
Status of Ribozyme and Antisense-Based Developmental Approaches for Anti-HIV-1 Therapya
Annals of the New York Academy of Sciences, 1992
The observation that the complementary interaction of nucleotide sequences exists in nature for the regulation of cellular functions led to the concept of "antisense" as a biological entity.' Antisense is any DNA or RNA that inhibits a gene function by methods that require complementary base-pairing to the genetic target. In the last 15 years, it has been shown that the expression of antisense RNA as well as the exogenous introduction of antisense DNA can affect a variety of cell function^.^-^ The observation that RNA has autocatalytic function that is mediated in part by complementary base-pairing has led to a second gcneration of antisense which is called ribozyme. Ribozyme is an RNA molecule having catalytic enzyme activity that cleaves RNA in a sequence-dependent manner, thereby inactivating the RNA. There are several types of ribozymes. (For a review see ref. 6 . The first of these, from the Tetrahymena group one intervening sequence, was shown by Zaug and Cech7 to be essential for cleaving an intron from messenger RNA. A second ribozyme was noted by Altmann and co-workers to function as the RNA component of RNAseP in the maturation of tRNAsX RNAseP consists of both RNA and protein moieties that function together to complete the 5' cleavage of pre-tRNAs. In this situation, the RNA component can function independently, but the 14-kD protein moiety is thought to create a microenvironment that optimizes the catalytic reaction . A third type of ribozyme, the hammerhead ribozyme, is the one that exists in certain viroids and virusoids as well as in Neurospora. These ribozymes require specific flanking sequences that target the ribozyme and bring together an active center, causing cleavage at that specific site. Synthetic ribozyme can be made synthetically, and the use of ribozyme for inhibition of HIV infection will be discussed in detail here.
Status of Ribozyme and Antisense-Based Developmental Approaches for Anti-HIV-1 Therapy
Annals of The New York Academy of Sciences - ANN N Y ACAD SCI, 1992
The observation that the complementary interaction of nucleotide sequences exists in nature for the regulation of cellular functions led to the concept of "antisense" as a biological entity.' Antisense is any DNA or RNA that inhibits a gene function by methods that require complementary base-pairing to the genetic target. In the last 15 years, it has been shown that the expression of antisense RNA as well as the exogenous introduction of antisense DNA can affect a variety of cell function^.^-^ The observation that RNA has autocatalytic function that is mediated in part by complementary base-pairing has led to a second gcneration of antisense which is called ribozyme. Ribozyme is an RNA molecule having catalytic enzyme activity that cleaves RNA in a sequence-dependent manner, thereby inactivating the RNA. There are several types of ribozymes. (For a review see ref. 6 . The first of these, from the Tetrahymena group one intervening sequence, was shown by Zaug and Cech7 to be essential for cleaving an intron from messenger RNA. A second ribozyme was noted by Altmann and co-workers to function as the RNA component of RNAseP in the maturation of tRNAsX RNAseP consists of both RNA and protein moieties that function together to complete the 5' cleavage of pre-tRNAs. In this situation, the RNA component can function independently, but the 14-kD protein moiety is thought to create a microenvironment that optimizes the catalytic reaction . A third type of ribozyme, the hammerhead ribozyme, is the one that exists in certain viroids and virusoids as well as in Neurospora. These ribozymes require specific flanking sequences that target the ribozyme and bring together an active center, causing cleavage at that specific site. Synthetic ribozyme can be made synthetically, and the use of ribozyme for inhibition of HIV infection will be discussed in detail here.
Effective Inhibition of Human Immunodeficiency Virus 1 Replication by Engineered RNase P Ribozyme
PLoS ONE, 2012
Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G 83-. U 83 and G 340-. A 340) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application.
Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells
Nature Medicine, 2009
Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system, and avoids lifetime highly active antiretroviral therapy. This study, the first randomized, double-blind, placebo-controlled, phase II cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1 infected adults who received a tat/vpr specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistical difference in viral load between the OZ1 and placebo group at the primary end-point (average at weeks 47 and 48) but time weighted areas under the curve from weeks 40-48 and 40-100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study provides the first indication that cell-delivered gene transfer is safe and biologically active in HIV patients and can be developed as a conventional therapeutic product.
Inhibition of HIV-1 Replication by an Improved Hairpin Ribozyme That Includes an RNA Decoy
RNA Biology, 2005
An anti-Tat hairpin ribozyme and a TAR RNA decoy were combined in one molecule. The chimeric molecule strongly inhibited HIV-1 replication (measured as changes in p24 levels in viral replication assays). The inhibitory action of the ribodecozyme (85%) was significantly greater than that shown by ribozyme and a non-catalytic variant carrying the functional decoy RNA domain (55% and 35%, respectively). This represents a significant improvement of the inhibitory efficiency of the ribozyme, suggesting there is an additive inhibitory effect on HIV-1 replication by the catalytic and decoy domains. This strategy could be used to create new inhibitor RNAs with enhanced in vivo performance.