Title- Post Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease �Journal- The New England Journal of Medicine.�Impact Factor- 176.0�Authors- David A. Williams ., etal���

Rithika R Nadar (21BIT31)

2^nd yr. MSC Biotechnology

SBT, MKU

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Introduction:

• Sickle cell disease is a common inherited disease affecting approximately 100,000 persons in the United States (Ware RE etal., 2010) worldwide about 400,000 infants are born with sickle cell disease each year.
• Sickle hemoglobin (HbS) is a variant of normal adult hemoglobin (HbA) that is produced when the β-globin gene (HBB) contains a single E6V missense mutation.
• On deoxygenation, HbS polymerizes, leading to abnormally shaped red cells and including painful Vaso occlusive crises, chronic hemolytic anemia, organ damage, decreased quality of life, and early death (Howes RE etal., 2013).

BACKGROUND:

• A high level of erythrocyte fetal hemoglobin (HbF) comprising α- and γ-globins may ameliorate these manifestations by mitigating sickle hemoglobin polymerization and erythrocyte sickling. BCL11A is a repressor of γ-globin expression and HbF production in adult erythrocytes. Its down-regulation is a promising therapeutic strategy for induction of HbF

Methods:

• The investigational therapy involved infusion of autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes a short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA (shmiR), allowing erythroid lineage–specific knockdown.
• Patients with an HLA matched sibling who could serve as donor for transplantation were excluded from the study.
• Apheresis for manufacture of the BCH-BB694 BCL11A shmiR drug product was performed, with a targeted collection for cell manufacturing of at least 4 million CD34+ cells per kilogram.
• CD34+ cells were selected, transduced with the BCH-BB694 BCL11A shmiR lentiviral vector and cryopreserved. Cell transduction was performed.
• Patients received fully myeloablative conditioning with intravenous busulfan for 4 consecutive days, with pharmacokinetic dose adjustment to target a daily area under the curve of approximately 5500 μM per minute.
• The transduced CD34+ cells were infused at least 24 hours after the last dose of busulfan.

Clinical, Imaging, and Laboratory Assessments:

• The clinical features of sickle cell anemia are compared qualitative, are based on data.
• Vector copy number was determined with the use of a (qPCR) assay performed.
• To determine the percentage of progenitor cells within the product that were transduced, clonogenic hematopoietic progenitors were plated in methylcellulose for 12 to 14 days, followed by qPCR assay of individual colonies to detect the integrated lentiviral vector.
• The blood samples were assessed for hemoglobin by blood counts, HbF by high-performance liquid chromatography.
• The clinical data were collected, including any reports of vaso-occlusive pain or other sickle-related events.

Patients & Treatment:

• Six patients (7 to 25 years of age at enrollment) received the gene therapy between February 2018 and March 2020, and the patients had a median follow-up of 18 months after infusion.
• The severe manifestations of sickle cell disease that qualified the patients for enrollment include stroke, Vaso-occlusive and priapism events.
• Patients were infused with the BCH-BB694 BCL11A shmiR drug product, which contained 4.9 million to 8.3 million CD34+ cells per kilogram of body weight. After the infusion all patients.

Laboratory Outcomes:

• After infusion with the BCH-BB694 BCL11A shmiR drug product, the HbF fraction of total Hb increased and then remained stable in all patients.
• Hemoglobin increased to a new baseline of 9.3 to 11.4 g per deciliter in the five untransfused patients and was stable over time. Hemolysis continued in all patients but was reduced from baseline, with the absolute reticulocyte count falling from 175,000– 680,000 per microliter.
• At the most recent study visit as of October 2020 (6 to 24 months after infusion), the median percentage of HbF as measured by high-performance liquid chromatography in the five untransfused patients was 30.4% .
• In all six patients, the induction of HbF was uniformly high, with a median HbF/ (HbF+HbS) of 30.5% The level of total HbF appeared to be stable. The induction of HbF was distributed broadly across erythrocytes. The median percentage of F-cells among untransfused red cells was 70.8%
• In patients 3, 4, 6, 7, and 8, HbF per F-cell increased Patient 2, had only 0.3% F-cells at baseline, At 24 months of follow-up. Patient 2 had 71% F-cells.
• The results indicate normal development and function of the B-cell compartment and suggest an absence of toxicity consistent with erythroid specific BCL11A suppression.

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Diagnosis:

• The treatment for sickle cell disease is allogeneic hematopoietic stem-cell transplantation. Matched sibling donor transplantation is curative in more than 90% of patients(Gluckman E etal., 2017, Bories D etal., 2020) but limitations include a higher risk of complications in older patients (Gluckman E etal., 2017) a risk of severe graft-versus-host disease (GVHD), and lack of an available matched sibling in approximately 80% of cases.
• In older children and adults, a higher level of HbF is associated with lower disease severity in sickle cell disease, has been observed in persons with hereditary persistence of HbF and in those who have a good response to hydroxyurea.
• Hydroxyurea leads to HbF induction in many patients, it is not effective in all patients.
• We have previously described a lentiviral vector that mediates potent erythroid-specific knockdown of BCL11A through RNA interference (RNAi), using a microRNA (miRNA)-adapted short hairpin RNA (shRNAmiR).(Brendel C etal., 2016, Guda S etal., 2015)

Result:

• As of October 2020, six patients had been followed for at least 6 months after receiving BCH-BB694 gene therapy; median follow-up was 18 months.
• All the patients who could be fully evaluated achieved robust and stable HbF induction (percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.3%), with HbF broadly distributed in red cells (F-cells 58.9 to 93.6% of untransfused red cells) and HbF per F-cell of 9.0 to 18.6 pg per cell.
• Clinical manifestations of sickle cell disease were reduced or absent during the follow-up period.

Discussion:

• We report findings in six patients with severe sickle cell disease treated with therapy directed against BCL11A, in this case through infusion with BCH-BB694 BCL11A shmiR transduced CD34+ cells, an approach of hemoglobin switching in order to increase intracellular HbF concentration and reduce HbS.
• All six had reached more than 6 months of follow-up as of October 2020 and had achieved robust induction of HbF. In transduced cells, BCL11A is suppressed by an shRNA embedded in a microRNA endogenous cellular machinery to produce a simultaneous increase in HbF protein and decrease in HbS protein.
• Altering the balance of intracellular globin production is particularly advantageous, because even a small decrease in HbS concentration results in a large increase in the time to polymer formation, the main determinant of the sickling process in red cells.

What is my perception to choose this topic?

• The use of a short hairpin RNA embedded within an endogenous microRNA scaffold, termed a shmiR vector, to alter genetic expression, rather than the addition of a protein coding sequence.
• This development has potential implications for other diseases that could benefit from down-regulation of gene expression rather than addition of a gene.
• Many other inherited related diseases are cured by gene silencing like thalassemia, cystic fibrosis and hemophilia, etc.
• Many crucial symptoms of sickle cell anemia have been cured by gene silencing use of microRNA.

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Conclusion:

• This study validates BCL11A inhibition as an effective target for HbF induction and provides preliminary evidence that shmiR-based gene knockdown offers a favorable risk–benefit profile in sickle cell disease.

Reference:

• John P Manis, M.D., David A. Williams, M.D., etal. The New England Journal of Medicine2021.,384;3.
• Heeney MM, Ware RE. Hydroxyurea for children with sickle cell disease. Hematol Oncol Clin North Am 2010;24:199-214.
• Piel FB, Patil AP, Howes RE, et al. Global epidemiology of sickle haemoglobin in neonates: a contemporary geostatistical model-based map and population estimates. Lancet 2013;381:142-51.
• Gluckman E, Cappelli B, Bernaudin F, et al. Sickle cell disease: an international survey of results of HLA-identical sibling hematopoietic stem cell transplantation. Blood 2017;129:1548-56.
• Bernaudin F, Dalle J-H, Bories D, et al. Long-term event-free survival, chimerism and fertility outcomes in 234 patients with sickle-cell anemia younger than 30 years after myeloablative conditioning and matched-sibling transplantation in France. Haematologica 2020;105:91-101.
• Brendel C, Guda S, Renella R, et al. Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype. J Clin Invest 2016;126:3868-78.
• Guda S, Brendel C, Renella R, et al. miRNA-Embedded shRNAs for lineagespecific BCL11A knockdown and hemoglobin F Induction. Mol Ther 2015;23: 1465-74.

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THANK YOU

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