Gene therapy to cure HIV infection.

To date, the only intervention that has cured HIV infection has been bone marrow transplants from HIV-resistant donors to HIV-infected recipients. This approach has been used to both cure hematological malignancies and HIV infection, but it cannot be widely adopted due to the high risk of mortality associated with cell transplants between individuals. To overcome this limitation, several approaches have been developed to generate HIV resistance using gene therapy in an infected individual's own cells. With the growing arsenal of effective methods to generate HIV-resistant cells, a safe and effective combination gene therapy approach to cure HIV infection is fast approaching. Here, we review several gene therapy-based methods to generate HIV-resistant cells including the expression of antiviral genes, genome editing, and transcriptional gene silencing. Their varied mechanisms, advantages, and disadvantages are discussed, and perspectives are provided for how they may be combined to design an effective gene therapy for HIV.

[Gene therapy to cure HIV infection]. / Thérapie génique pour guérir l'infection par le VIH.

To date, the only intervention that has cured HIV infection has been bone marrow transplants from HIV-resistant donors to HIV-infected recipients. This approach has been used to both cure hematological malignancies and HIV infection, but it cannot be widely adopted due to the high risk of mortality associated with cell transplants between individuals. To overcome this limitation, several approaches have been developed to generate HIV resistance using gene therapy in an infected individual's own cells. With the growing arsenal of effective methods to generate HIV-resistant cells, a safe and effective combination gene therapy approach to cure HIV infection is fast approaching. Here, we review several gene therapy-based methods to generate HIV-resistant cells including the expression of antiviral genes, genome editing, and transcriptional gene silencing. Their varied mechanisms, advantages, and disadvantages are discussed, and perspectives are provided for how they may be combined to design an effective gene therapy for HIV.

RNA structure modulates Cas13 activity and enables mismatch detection.

The RNA-targeting CRISPR nuclease Cas13 has emerged as a powerful tool for applications ranging from nucleic acid detection to transcriptome engineering and RNA imaging1-6. Cas13 is activated by the hybridization of a CRISPR RNA (crRNA) to a complementary single-stranded RNA (ssRNA) protospacer in a target RNA1,7. Though Cas13 is not activated by double-stranded RNA (dsRNA) in vitro, it paradoxically demonstrates robust RNA targeting in environments where the vast majority of RNAs are highly structured2,8. Understanding Cas13's mechanism of binding and activation will be key to improving its ability to detect and perturb RNA; however, the mechanism by which Cas13 binds structured RNAs remains unknown9. Here, we systematically probe the mechanism of LwaCas13a activation in response to RNA structure perturbations using a massively multiplexed screen. We find that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: structure in the protospacer region competes with the crRNA and can be disrupted via a strand-displacement mechanism, while structure in the region 3' to the protospacer has an allosteric inhibitory effect. We leverage the kinetic nature of the strand displacement process to improve Cas13-based RNA detection, enhancing mismatch discrimination by up to 50-fold and enabling sequence-agnostic mutation identification at low (<1%) allele frequencies. Our work sets a new standard for CRISPR-based nucleic acid detection and will enable intelligent and secondary-structure-guided target selection while also expanding the range of RNAs available for targeting with Cas13.