Whittaker, Thomas E;
Moula, Shefta E;
Bahal, Sameer;
Bakri, Faris G;
Hayajneh, Wail A;
Daoud, Ammar K;
Naseem, Asma;
... Santilli, Giorgia; + view all
(2023)
Multidimensional Response Surface Methodology for the development of a gene editing protocol for p67phox-deficient Chronic Granulomatous Disease.
Human Gene Therapy
10.1089/hum.2023.114.
(In press).
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Abstract
Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using CRISPR/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34+ hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimisation process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach to optimisation of gene correction to rapidly investigate and select editing conditions for CD34+ cells with the best possible balance between correction and cell/CFU loss in a parsimonious one-shot experiment. This method revealed that using low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34+ cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimised editing conditions for the correction of p67phox-deficient chronic granulomatous disease, an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections, allowing rescue of p67phox expression and functional correction of CD34+-derived neutrophils from a CGD patient.
| Type: | Article |
|---|---|
| Title: | Multidimensional Response Surface Methodology for the development of a gene editing protocol for p67phox-deficient Chronic Granulomatous Disease |
| Location: | United States |
| DOI: | 10.1089/hum.2023.114 |
| Publisher version: | https://doi.org/10.1089/hum.2023.114 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Infection, Immunity and Inflammation Dept |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10183775 |
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