Multivalent anti-CCR ribozymes for stem cell-based HIV type 1 gene therapy

Area of Science:

• Virology
• Gene Therapy
• Molecular Biology

Background:

• HIV-1 entry relies on CD4 receptors and CCR5/CXCR4 coreceptors.
• CCR5 genetic defects confer resistance to HIV-1, highlighting CCR5 as a therapeutic target.
• Previous ribozyme studies showed potential for CCR5 downregulation.

Purpose of the Study:

• To enhance CCR5 downregulation using a novel anti-CCR5 ribozyme heterotrimer (R5RbzTM).
• To assess the efficacy of R5RbzTM in reducing HIV-1 infection in vitro and in gene-modified cells.
• To evaluate the safety and efficacy of R5RbzTM in hematopoietic progenitor cells for gene therapy.

Main Methods:

• Designed and synthesized an anti-CCR5 ribozyme heterotrimer targeting three CCR5 mRNA cleavage sites.
• Utilized retroviral vectors for stable gene transduction of R5RbzTM into HOS.CD4/R5 cells and CD34+ hematopoietic progenitor cells.
• Assessed CCR5 surface expression, viral infectivity, and hematopoietic progenitor cell differentiation post-transduction.

Main Results:

• In vitro tests confirmed R5RbzTM's ability to cleave CCR5 RNA substrates.
• Transduction of HOS.CD4/R5 cells with R5RbzTM led to reduced CCR5 expression and 70% less macrophage-tropic HIV-1 infection.
• Transduced CD34+ hematopoietic progenitor cells differentiated normally and exhibited resistance to M-tropic HIV-1 infection.

Conclusions:

• The anti-CCR5 ribozyme heterotrimer is effective in downregulating CCR5 expression and conferring resistance to HIV-1 infection.
• This multivalent ribozyme approach is suitable for gene therapy applications in HIV treatment.
• The study supports the use of trimeric anti-CCR5 ribozymes for developing novel HIV gene therapies.