Selection of RNAi-based inhibitors for anti-HIV gene therapy

Knoepfel, Stefanie A; Centlivre, Mireille; Liu, Ying Poi; Boutimah, Fatima; Berkhout, Ben

doi:10.5501/wjv.v1.i3.79

Cited by 15 publications

(9 citation statements)

References 70 publications

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“…Here, we selected four anti-HIV-1 shRNA candidates that target essential HIV-1 genes and highly conserved regions of the HIV-1 genome among all HIV-1 subtypes (>73% target conservation of the shRNA target sequence in all virus isolates present in the Los Alamos database), in particular subtype B isolates (>80%). 30 We evaluated the preclinical safety profile in vivo in view of further development of this RNAi-based gene therapy towards clinical trial phase I.…”

Section: Discussionmentioning

confidence: 99%

Preclinical In Vivo Evaluation of the Safety of a Multi-shRNA-based Gene Therapy Against HIV-1

Centlivre

Legrand

Klamer

et al. 2013

Molecular Therapy - Nucleic Acids

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Highly active antiretroviral therapy (HAART) has significantly improved the quality of life and the life expectancy of HIV-infected individuals. Still, drug-induced side effects and emergence of drug-resistant viral variants remain important issues that justify the exploration of alternative therapeutic options. One strategy consists of a gene therapy based on RNA interference to induce the sequence-specific degradation of the HIV-1 RNA genome. We have selected four potent short hairpin RNA (shRNA) candidates targeting the viral capside, integrase, protease and tat/rev open-reading frames and screened the safety of them during human hematopoietic cell development, both in vitro and in vivo. Although the four shRNA candidates appeared to be safe in vitro, one shRNA candidate impaired the in vivo development of the human immune system in Balb/c Rag2−/−IL-2Rγc−/− (BRG) mice. The three remaining shRNA candidates were combined into one single lentiviral vector (LV), and safety of the shRNA combination during human hematopoietic cell development was confirmed. Overall, we demonstrate here the preclinical in vivo safety of a LV expressing three shRNAs against HIV-1, which is proposed for a future Phase I clinical trial.

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Section: Discussionmentioning

confidence: 99%

Preclinical In Vivo Evaluation of the Safety of a Multi-shRNA-based Gene Therapy Against HIV-1

Centlivre

Legrand

Klamer

et al. 2013

Molecular Therapy - Nucleic Acids

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“…A negative impact of a single shRNA on in vitro cell growth and hematopoiesis in the human immune system mouse was observed, which led to its removal from the translational track toward a clinical trial. 22,23 We, therefore, created the combinatorial RNAi vector R3A that expresses the three nontoxic and potent antiviral shRNAs: Pol1, Pol47, and RT5.…”

Section: Introductionmentioning

confidence: 99%

The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS

2014

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RNA interference (RNAi) is highly effective in inhibiting human immunodeficiency virus type 1 (HIV-1) replication by the expression of antiviral short hairpin RNA (shRNA) in stably transduced T-cell lines. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against highly conserved regions of the HIV-1 RNA genome. The future in vivo application of such a gene therapy protocol will reach only a fraction of the T cells, such that HIV-1 replication will continue in the unmodified T cells, thereby possibly frustrating the therapy by generation of HIV-1 variants that escape from the inhibition imposed by the protected cells. We studied virus inhibition and evolution in pure cultures of shRNA-expressing cells versus mixed cell cultures of protected and unprotected T cells. The addition of the unprotected T cells indeed seems to accelerate HIV-1 evolution and escape from a single shRNA inhibitor. However, expression of three antiviral shRNAs from a single lentiviral vector prevents virus escape even in the presence of unprotected cells. These results support the idea to validate the therapeutic potential of this anti-HIV approach in appropriate in vivo models.

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“…For instance, antiviral gene therapy based on the mechanism of RNA interference (RNAi) uses short hairpin RNA (shRNA) inhibitors that target the viral RNA genome 8 , 9 . We previously demonstrated that RNAi attack is influenced by the local RNA structure of the HIV-1 RNA target sequence 10 , 11 and more recently reported that the design of shRNA inhibitors can be significantly improved by the exclusive targeting of accessible HIV-1 RNA domains as determined by SHAPE technology 12 …”

Section: Introductionmentioning

confidence: 99%

On the role of four small hairpins in the HIV-1 RNA genome

Knoepfel

Berkhout

2013

RNA Biology

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An RNA secondary structure model for the complete HIV-1 genome has recently been published based on SHAPE technology. Several well-known RNA motifs such as TAR and RRE were confirmed and numerous new structured motifs were described that may play important roles in virus replication. The 9 kb viral RNA genome is densely packed with many RNA hairpin motifs and the collective fold may play an important role in HIV-1 biology. We initially focused on 16 RNA hairpin motifs scattered along the viral genome. We considered conservation of these structures, despite sequence variation among virus isolates, as a first indication for a significant function. Four relatively small hairpins exhibited considerable structural conservation and were selected for experimental validation in virus replication assays. Mutations were introduced into the HIV-1 RNA genome to destabilize individual RNA structures without affecting the protein-coding properties (silent codon changes). No major virus replication defects were scored, suggesting that these four hairpin structures do not play essential roles in HIV-1 replication.

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Selection of RNAi-based inhibitors for anti-HIV gene therapy

Cited by 15 publications

References 70 publications

Preclinical In Vivo Evaluation of the Safety of a Multi-shRNA-based Gene Therapy Against HIV-1

Preclinical In Vivo Evaluation of the Safety of a Multi-shRNA-based Gene Therapy Against HIV-1

The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS

On the role of four small hairpins in the HIV-1 RNA genome

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