# Rectifying splicing mutations in blood disorders by gene editing

> **NIH NIH R01** · BOSTON CHILDREN'S HOSPITAL · 2022 · $861,736

## Abstract

Inherited  blood disorders are especially favorable  targets for therapeutic  genome editing in 
that ex vivo modification of patient hematopoietic  stem cells (HSCs) followed by autologous  
transplantation can result in lifelong  recovery of normal blood cell production. Recently we 
developed  an improved version of SpCas9 (3xNLS-SpCas9) and an efficient electroporation protocol 
for genome editing  of CD34+ hematopoietic  stem and progenitor  cells (HSPCs) using SpCas9 
ribonucleoprotein (RNP) that leads to highly efficient on-target gene modification, preservation of 
HSC function and undetectable  off-target editing.
In principle , homologous recombination (HR) or base editing  could be harnessed for the precise 
correction of disease-associated mutations. However, the requirement  for co-delivery of donor 
template sequence, the cell cycle dependence of HR-based gene repair, and the competing 
nonhomologous  end­ joining/microhomology mediated end joining  mutagenic repair pathways 
complicate achieving efficient HR in HSCs. Base editing Is currently limited in its targeting range 
with uncertainty about potential  genotoxicity  and HSC efficiency. Nuclease-induced predictable 
end-joining  repair (with indels) is a highly efficient means of  gene modification, and could 
itself be therapeutic depending  on the allelic outcome. This strategy may be particularly  
effective for noncoding mutations that impact regulatory  elements, such as those that dictate the 
pattern of mRNA splicing. We hypothesize that genome editing, by directing efficient non-templated  
end-joining DNA repair in HSCs, could restore gene expression and provide durable therapy for 
inherited  blood disorders associated with splicing mutations.
Two of the most common mutations associated with transfusion-dependent β-thalassemia are HBB IVS1- 
11OG>A and IVS2-654C> T which introduce intronic  aberrant splice acceptor and donor sites 
respectively.
Using SpCas9 and LbCas12a RNPs, we have successfully disrupted these inappropriate  regulatory 
elements in HSPCs from multiple patient donors. The erythrocytes differentiated  in vitro from 
these nuclease-treated cells display robust increase in normally spliced HBB mRNA and restored 
adult hemoglobin  (HbA)  expression, suggesting that this is a potent strategy for therapeutic 
development.  In Aims 1 & 2 we will develop  Cas9 and Cas12a editing reagents for these splicing 
mutations through nuclease optimization, unbiased genome-wide off-target  analysis, and assessment 
of HSC editing  rates through xenoengraftment of edited  β-thalassemia patient HSPCs. In Aim 3, we 
will develop efficient strategies for the non-templated  gene editing repair of splice junction 
disrupting mutations for the IVS2+2T>C mutation in SBOS commonly associated with Shwachman­
Diamond syndrome. The successful completion of these studies w/1 define editing approaches  for the 
efficient HSC repair of a range of pathogenic  splicing mutations that...

## Key facts

- **NIH application ID:** 10305646
- **Project number:** 5R01HL150669-03
- **Recipient organization:** BOSTON CHILDREN'S HOSPITAL
- **Principal Investigator:** Daniel Evan Bauer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $861,736
- **Award type:** 5
- **Project period:** 2019-12-20 → 2023-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10305646

## Citation

> US National Institutes of Health, RePORTER application 10305646, Rectifying splicing mutations in blood disorders by gene editing (5R01HL150669-03). Retrieved via AI Analytics 2026-06-14 from https://api.ai-analytics.org/grant/nih/10305646. Licensed CC0.

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