Sickle cell disease is a genetic disorder that results in the production of a dysfunctional form of hemoglobin. In the United States about 100,000 people have sickle cell disease and worldwide almost 300,000 affected children are born every year. b-thalassemia is also an inherited disorder that results in the decreased synthesis or complete absence of the b-globin chains of hemoglobin. The estimated annual incidence of b-thalassemia is 500,000 most commonly to parents from Mediterranean countries, North Africa, the Middle East, India, Central Asia, and Southeast Asia. Both diseases are caused by mutations in the b-globin (HBB) gene and can be cured by allogeneic hematopoietic stem cell transplantation (allo-HSCT or provocatively called ?allogeneic gene therapy?). The lack of available immunologically matched donors and the significant morbid complications from allo-HSCT, however, means that allogeneic gene therapy has only been used to treat a small fraction of the patients who could benefit. There are currently no other definitive and curative approaches for either of these diseases. We have developed an alternative approach that has the potential to circumvent all of these issues described. For sickle cell disease, the functional gene correction process we are developing uses CRISPR/Cas9 to edit the HBB gene directly in patient?s own HSPCs by precisely correcting the sickling point mutation. But b-thalassemia is caused by mutations throughout the gene. For this we will either knock-in a wild-type cDNA into exon 1 of the HBB gene such that the cDNA utilizes the endogenous ATG initiation codon or knock-in a full length HBB gene into HBA1. In this way the HBB gene will be expressed using the endogenous initiation start codon and using all of the endogenous natural regulatory elements. By knocking into HBA1 we will also simultaneously create a-thalassemia trait, a genotype that is known to decrease the severity of b-thalassemia.
In Aim 1 - we will correct the sickle mutation in the endogenous HBB gene using genome editing;
in Aim 2 we focus on developing the knock-in strategy to allow expression of the HBB cDNA in HSPCs from b-thalassemia patients.
The final aim (Aim 3) is to use a series of functional assays to test the overall toxicity of the optimized process and thereby determine the safety of the genome editing process. As part of this grant we have assembled a team of co-Investigators and consultants with expertise in creating a GMP compatible cell manufacturing process. Collectively our goal is to develop reagents that optimize HBB gene correction thereby providing key IND enabling data to move forward to first-in-human clinical phase I/II clinical trials for the b-hemoglobinopathies. !

Public Health Relevance

Sickle cell disease and b-thalassemia are caused by inherited mutations in the b-globin (HBB) gene and afflict millions of people worldwide. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can cure these diseases, but the lack of immunologically matched donors and toxic side effects means this strategy has only treated a small fraction of patients. This proposal will develop our existing CRISPR/Cas9 genome editing strategy to create a population of autologous HBB corrected HSPCs that can confer stable long-term cure of patients with sickle cell disease and b-thalassemia.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Therapeutic Approaches to Genetic Diseases Study Section (TAG)
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Qasba, Pankaj
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Stanford University
Schools of Medicine
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