USE OF PRIME EDITING FOR TREATING SICKLE CELL DISEASE

Smart overview of the Invention

The disclosure outlines a method for treating sickle cell disease (SCD) using prime editing technology. This approach involves the use of engineered prime editing guide RNAs (epegRNAs) specifically targeting the β-globin (HBB) gene. The prime editing system offers a way to correct the genetic mutation responsible for SCD by directly altering the DNA sequence without the need for double-stranded breaks, which are common in other gene-editing techniques.

Background

Sickle cell disease is a genetic disorder caused by a single nucleotide mutation in the HBB gene, leading to the production of abnormal hemoglobin. This mutation results in the formation of sickle-shaped red blood cells, causing various health complications. Current treatments, such as hematopoietic stem cell transplantation, are limited by donor compatibility issues and potential side effects. There is a need for methods that can correct the genetic mutation without causing additional genomic damage.

Prime Editing Strategy

Prime editing allows precise DNA modifications by replacing a target DNA segment with a new sequence. The strategy described in the disclosure corrects the SCD mutation back to the normal HBB allele with high efficiency and minimal off-target effects. In experiments, human hematopoietic stem cells (HSPCs) edited using this method showed sustained correction levels and reduced sickling. The approach demonstrates a potential one-time treatment for SCD, avoiding the complications of other gene-editing methods.

Methodology

The method involves using a prime editor and epegRNA to target the specific mutation in the HBB gene. The epegRNA includes a spacer sequence, sgRNA scaffold, and other structural components to guide the editing process. The prime editing system can also introduce a silent PAM-disrupting edit to prevent reversion of the corrected sequence. Additionally, nicking sgRNA may be used to enhance the incorporation of edits by cellular repair mechanisms.

Application and Delivery

The prime editing components can be delivered to cells, such as human hematopoietic stem or progenitor cells, via electroporation. This method is particularly suited for ex vivo editing, where edited cells are transplanted back into the patient. The edited cells maintain the introduced edits in their sub-lineages, providing a durable treatment effect. The approach offers a promising avenue for treating SCD with potentially fewer risks compared to existing therapies.