# Amelioration of Beta-hemoglobinopathies by efficient precise deletion of the +58 BCL11A enhancer using orthogonal Cas9-Cas9 chimeras

> **NIH NIH F31** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2020 · $32,833

## Abstract

Project Summary
β-Thalassemia and sickle cell disease (SCD) are severe inherited anemias that result in defective β-globin
expression. A common feature shared between both disorders is that the diseases can be mitigated by the
production of fetal hemoglobin (HbF). Current treatments for both disorders are mainly supportive and focus on
alleviating disease complications. However, management of these β-hemoglobinopathies is expensive,
restrictive, lifelong, and associated with significant side effects. Currently, allogeneic hematopoietic stem cell
therapy is the only curative treatment for β-hemoglobinopathies. However, finding human leukocyte antigen
(HLA)-matched donors is highly challenging. Gene editing approaches that alter the expression of HbF should
improve the health and quality of life of individuals with β-hemoglobinopathies.
 The goal of this project is to develop efficient, accurate, and safe CRISPR gene editing approaches as
a universal treatment for β-hemoglobinopathies. Typically, a nuclease (SpyCas9) and guide RNA (gRNA)
target a specific region (+58 enhancer of BCL11A) and creates a double-stranded break. Small insertions or
deletions (InDels) are created when imprecise repair of the DNA break occurs, resulting in inactivation of the
target gene or functional element. Current therapeutic editing approaches for β-hemoglobinopathies at the
BCL11A locus focus on the mutagenesis of a single GATA1 recognition sequence within the +58 enhancer in
CD34+ hematopoietic stem and progenitor cells (HSPCs), limiting the scope of their potential impact.
 This project will harness our recently developed Cas9-Cas9 fusion chimera, which is able to produce
segmental deletions with defined junctions (precise deletions) at a higher efficiency than standard Cas9
nucleases. Furthermore, Cas9-Cas9 fusions are able to effectively target suboptimal PAM sequences, and
thus these nucleases have a broader targeting range than standard SpyCas9. These properties make Cas9-
Cas9 fusions ideal for the deletion of therapeutically relevant regulatory elements, such as the +58 enhancer of
BCL11A. Our preliminary studies show that ribonucleoprotein (RNP) complexes of these Cas9-Cas9 fusions
targeting this locus are functional in CD34+ HSPCs and result in robust induction of HbF.
 In Aim 1, I will define deletion products within the +58 enhancer that facilitate the maximum induction of
HbF in erythroid model systems and optimize the Cas9-Cas9 fusions to efficiently and specifically produce
these precise deletions with minimal collateral damage to the genome. In Aim 2, optimized Cas9-Cas9 fusion
protein-sgRNA complexes will be delivered into CD34+ HSPCs and HbF induction levels will be quantified in
erythroid progeny. Treated CD34+ cells will be engrafted into immunodeficient mice to measure engraftment
potential and persistence of editing in long-term hematopoietic stem cells (LT-HSCs). The genome-editing
tools generated from the proposed work will provide a path to i...

## Key facts

- **NIH application ID:** 9959197
- **Project number:** 5F31HL147482-02
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** Kevin Luk
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $32,833
- **Award type:** 5
- **Project period:** 2019-05-09 → 2021-05-08

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9959197

## Citation

> US National Institutes of Health, RePORTER application 9959197, Amelioration of Beta-hemoglobinopathies by efficient precise deletion of the +58 BCL11A enhancer using orthogonal Cas9-Cas9 chimeras (5F31HL147482-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/9959197. Licensed CC0.

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