Activation of the Protective Arm of Renin Angiotensin System (RAS) Corrects the Reparative Dysfunction of Diabetic CD34+ Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2637-2637
Author(s):  
Yagna PR Jarajapu ◽  
Sergio Caballero ◽  
Li Liu ◽  
Vinayak Shenoy ◽  
Michael J Katovich ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Surface Expression ◽  
Receptor Expression ◽  
Nadph Oxidase Activity ◽  
Mas Receptor ◽  
Autologous Cell ◽  
Cd34 Cells ◽  
Autologous Cell Therapy ◽  
No Bioavailability

Abstract Abstract 2637 Purpose: RAS plays a vital role in regulating many physiological processes of the vascular system. Angiotensin II (Ang II), a product of angiotensin converting enzyme (ACE), mediates its effects through activation of either the AT1 receptor – to induce vasoconstriction, proliferation, fibrosis, and inflammation – or the AT2 receptor to promote NO generation. The protective arm of RAS involves ACE2, which produces angiotensin-(1-7) [Ang-(1-7)]. Ang-(1-7) activates the MAS receptor to promote vascular health. Because diabetic endothelial progenitor cells are dysfunctional and this limits their utility in autologous cell therapy, we asked whether angiotensin (Ang)-(1-7) could restore the vasoreparative function of diabetic CD34+cells. Methods: Healthy nondiabetic (ND) and diabetic (D) Lin−CD45midCD34+ cells were obtained from peripheral blood mononuclear cells (PB-MNCs) by FACS. The effect of Ang-(1-7) on migration, proliferation, NO bioavailability, reactive oxygen species (ROS) levels and NADPH oxidase activity were evaluated in ND- and D-CD34+ cells. The effect of Ang-(1-7) on the formation of ECFCs from ND- and D-MNCs was evaluated. Ang-(1-7) production by cells was analyzed and the expression of ACE2 and Mas-receptor were assessed by real-time PCR and flow cytometry. Effects of ACE2 activators XNT and DIZE in CD34+ cells were also evaluated. D-CD34+ cells were genetically modified to overexpress Ang-(1-7) by lentiviral Ang-(1-7)-fusion transgene and their function was evaluated in vitro. The effect of transduction on the surface expression of CD133, CD34 and CD309 was assessed. In vivo homing function was assessed in a mouse model of ischemia-reperfusion (I/R). After one week of I/R insult, when retinal capillary damage was appreciable, CD34 cells were intravitreally injected. Neural retinas were harvested after 48 hours and human cells within the mouse vasculature were localized by immunohistochemistry. Results: Migration to SDF1- and VEGF-were impaired in D-CD34+ cells. In contrast, Ang-(1-7)-induced migration in both D-CD34+ and ND-CD34+cells was dependent on Mas receptor expression and eNOS. ROS levels and NADPH oxidase activity were reduced and proliferation and NO bioavailability were restored in D-CD34+ cells by Ang-(1-7). ECFCs from D-MNCs were appeared only in the presence of Ang-(1-7). Migration, NO release and Ang-(1-7) release by ACE2 activators, XNT and DIZE, were significantly decreased in D-CD34+ cells. Ang-(1-7) release and ACE2 expression were decreased in D-cells while Mas-receptor expression was similar that in ND-cells. Ang-(1-7) gene-modified cells showed reduced ROS levels, increased NO bioavailability, enhanced migration to SDF-1 and proliferation. Lentiviral transduction did not alter surface expression of CD133, CD34 and CD309. Ang-(1-7)-overexpression restored the homing efficiency of D-CD34+ cells similar to that of ND-CD34+ cells in vivo. Conclusions: Ang-(1-7) stimulates the vasoreparative functions in CD34+ cells. Ang-(1-7) bypasses the reduced ACE2 seen in diabetic CD34+ cells and restores the vasoreparative potential of these cells by decreasing oxidative stress and normalizing NO bioavailability. Pharmacological strategies that either increase ACE2 or Ang-(1-7) in diabetic CD34+ cells will improve their therapeutic utility for autologous cell therapy in treatment of diabetic complications. Disclosures: No relevant conflicts of interest to declare

EDIT-301: An Experimental Autologous Cell Therapy Comprising Cas12a-RNP Modified mPB-CD34+ Cells for the Potential Treatment of SCD

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4636-4636
Author(s):  
Edouard De Dreuzy ◽  
Jack Heath ◽  
John A Zuris ◽  
Patricia Sousa ◽  
Ramya Viswanathan ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Fetal Hemoglobin ◽  
Employment Equity ◽  
Autologous Cell ◽  
Cd34 Cells ◽  
Autologous Cell Therapy ◽  
Equity Ownership ◽  
Ccaat Box

Sickle cell disease (SCD) is an inherited blood disorder affecting approximately 100,000 individuals in the United States. Fetal hemoglobin (HbF) is a major modifier of SCD severity. Studies showed that individuals with compound heterozygosity for sickle hemoglobin (HbS) and hereditary persistence of fetal hemoglobin (HPFH) that expressed approximately 30% HbF did not show features of SCD. Therefore, we are developing EDIT-301, an experimental autologous cell therapy comprising CD34+ cells genetically modified using a Cas12a RNP (ribonucleoprotein) to promote HbF expression to treat SCD. Several HPFH mutations have been reported at the HBG locus. In particular, disruption of the distal CCAAT-box region of the HBG1/2 promoters was associated with elevated levels of HbF, suggesting that this region is a relevant genome editing target for the treatment of SCD. Genotype to phenotype analysis at the distal CCAAT-box region of the HBG1/2 promoters identified the mutations leading to elevated HbF expression. Indels disrupting more than 3 nucleotides were generally associated with elevated HbF expression while smaller indels had lower to no impact. We evaluated editing at this site using several RNP configurations, based on either SpCas9 or Cas12a (also known as Cpf1). Cas12a RNP resulted in larger deletions and a higher frequency of productive indels than SpCas9 RNP. Furthermore, productive indels generated with SpCas9 RNP relied predominantly on microhomology mediated end joining (MMEJ) mechanism. As the MMEJ repair mechanism is not frequently used by hematopoietic stem cells (HSC), productive editing may be low in vivo. In contrast, Cas12a RNP generated more productive indels at the HBG1/2 promoters target site irrespective of the DNA repair mechanism. We therefore postulated that editing at the distal CCAAT-box region with Cas12a would better support long-term persistence of productive indels and robust HbF expression. Consistent with this hypothesis, 80-90% editing was observed after electroporation of mobilized peripheral blood CD34+ cells (mPB-CD34+ cells) from healthy donors with a Cas12a RNP targeting the HBG1/2 promoters, resulting in approximately 40% of HbF expression in their erythroid progeny. Infusion of the modified CD34+ cells into NBSGW mice resulted in long-term multi-lineage reconstitution at 16 weeks post-infusion, with no reduction in editing levels compared to those at the time of infusion. A diverse on-target editing profile was observed in all animals, indicative of a polyclonal engraftment. Erythroid cells purified from the bone marrow of animals that received Cas12a-RNP treated cells demonstrated robust HbF expression averaging 40-50% compared to ~5% observed in the vehicle-treated control group. Off-target activity of the Cas12a RNP was also evaluated. A set of candidate off-target sites was first determined using orthogonal methods, including: in-silico prediction, Digenome-Seq and GUIDE-Seq. Each candidate site was then analyzed for editing by targeted PCR-NGS in electroporated CD34+ cells. No off-target editing was verified, demonstrating the specificity of this Cas12a RNP. Taken together, we identified a specific Cas12a RNP that efficiently edited the distal CCAAT-box region at the HBG1/2 promoters in CD34+ cells. These edited CD34+ cells led to long-term polyclonal multilineage engraftment and therapeutically meaningful levels of 40-50% HbF in vivo. Based on these results, IND-enabling activities have been initiated for EDIT-301: an experimental autologous cell therapy comprising Cas12a-RNP modified mPB-CD34+ cells for the potential treatment of SCD. Disclosures De Dreuzy: Editas Medicine Inc.: Employment, Equity Ownership. Heath:Editas Medicine Inc.: Employment, Equity Ownership. Zuris:Editas Medicine Inc.: Employment, Equity Ownership. Sousa:Editas Medicine Inc.: Employment, Research Funding. Viswanathan:Editas Medicine Inc.: Employment, Equity Ownership. Scott:Editas Medicine Inc.: Employment, Equity Ownership. Da Silva:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Capehart:Editas Medicine Inc.: Equity Ownership. Wang:Editas Medicine Inc.: Employment, Equity Ownership. Fernandez:Editas Medicine Inc.: Employment, Equity Ownership. Myer:Editas Medicine Inc.: Employment, Equity Ownership. Albright:Editas Medicine Inc.: Employment, Equity Ownership. Wilson:Editas Medicine Inc.: Employment, Equity Ownership. Teixeira:Editas Medicine Inc.: Employment, Equity Ownership. Chang:Editas Medicine Inc.: Employment, Equity Ownership.

scholarly journals Robust Pre-Clinical Results and Large-Scale Manufacturing Process for Edit-301: An Autologous Cell Therapy for the Potential Treatment of SCD

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 45-46
Author(s):  
Edouard De Dreuzy ◽  
Jack Heath ◽  
Patricia Sousa ◽  
Tusneem Janoudi ◽  
Harry An ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Large Scale ◽  
Fetal Hemoglobin ◽  
Normal Donor ◽  
Publicly Traded ◽  
Autologous Cell ◽  
Cd34 Cells ◽  
Hbf Induction ◽  
Autologous Cell Therapy

Sickle cell disease (SCD) is an inherited blood disorder affecting approximately 100,000 individuals in the United States. As fetal hemoglobin (HbF) has been shown to be protective against clinical manifestation of SCD, we are developing EDIT-301, an autologous cell therapy comprising CD34+ cells genetically modified using a Cas12a ribonucleoprotein (RNP) to promote HbF expression to treat SCD. Fetal hemoglobin induction for EDIT-301 is achieved by disrupting the HBG1 and HBG2 promoter distal CCAAT-box region where naturally occurring mutations are found to be associated with elevated HbF expression. Cas12a was selected over Cas9 due to the more productive and sustainable (NHEJ derived) indel profile, as well as high specificity. Using Cas12a RNP, on-target editing of ~90% was achieved in mobilized peripheral blood CD34+ cells (mPB-CD34+ cells) from both healthy and SCD donors at research scale with no detectable off targets. Editing of CD34+ cells led to an average of 43% and 54% of HbF expression in the erythroid progeny of normal donor and SCD donor cells respectively in a pancellular fashion (~93% population). The robust HbF induction in SCD red blood cells (RBCs) resulted in significant phenotypic and functional improvement including reduced sickling and increased deformability under hypoxia ex vivo. Using a microfluidic assay that replicated blood flow in microvasculature under varying oxygen conditions, SCD RBCs derived from RNP electroporated CD34+ cells showed improved rheological behavior. The rheology improvement under hypoxia was strongly correlated with the increased levels of HbF in each sample. Infusion of the modified CD34+ cells from normal donors into NBSGW mice resulted in long-term multi lineage and polyclonal reconstitution. Editing levels at 16 weeks post infusion were > 90% in all human lineages tested, demonstrating the efficient editing of SCID-repopulating hematopoietic stem cells (HSCs). Consistent with the high editing levels, human erythroid cells from the bone marrow of mice that received Cas12a-RNP treated cells demonstrated pancellular (~90% F+ RBCs) HbF expression averaging 40-50% of total hemoglobin compared to ~5% HbF observed in the control group. We have developed a consistent large-scale process using functionally closed, semi-automated systems suitable for use in clinical manufacturing. We have shown robust editing of normal donor CD34+ cells and SCID-repopulating HSCs with the clinical scale process. Editing levels of >90% were detected after long term engraftment in mice. In summary, we have demonstrated successful on-target editing of mPB CD34+ cells derived from both normal and SCD donors using a Cas12a RNP, which coincided with robust HbF induction and a phenotypic reduction of sickling in the SCD erythroid progeny, as well as improved rheological behavior. Editing of the HBG1 and HBG2 promoters using this RNP was highly specific with no measurable off-target. In vivo, cells from normal donors readily engrafted and reconstituted all blood cell lineages at levels comparable to unedited cells. Finally, a robust large-scale manufacturing process has been developed to supply material for the clinical setting. Based on these results, we are completing the activities required to assess EDIT-301 in the clinic as treatment for SCD. Disclosures De Dreuzy: Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Heath:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Sousa:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Janoudi:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. An:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Albright:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Teixeira:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Monesmith:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Chang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company.

Characterization of Insulin-Secreting Porcine Bone Marrow Stromal Cells Ex Vivo and Autologous Cell Therapy in Vivo

2015 ◽  
Vol 24 (7) ◽  
pp. 1205-1220 ◽  
Author(s):  
Hai Van Thi Do ◽  
Wan Ting Loke ◽  
Irene Kee ◽  
Vivienne Liang ◽  
Sebastian J. David ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Therapy ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Ex Vivo ◽  
Marrow Stromal Cells ◽  
Autologous Cell ◽  
Autologous Cell Therapy

scholarly journals Protocol and Baseline Data on Renal Autologous Cell Therapy Injection in Adults with Chronic Kidney Disease Secondary to Congenital Anomalies of the Kidney and Urinary Tract

2021 ◽  
pp. 1-6
Author(s):  
Joseph Stavas ◽  
Maria Diaz-Gonzalez de Ferris ◽  
Ashley Johns ◽  
Deepak Jain ◽  
Tim Bertram
Keyword(s):  
Chronic Kidney Disease ◽  
Renal Function ◽  
Urinary Tract ◽  
Kidney Disease ◽  
Cell Therapy ◽  
Congenital Anomalies ◽  
Preliminary Analysis ◽  
Autologous Cell ◽  
Tissue Acquisition ◽  
Autologous Cell Therapy

Background: Advanced cell therapies with autologous, homologous cells show promise to affect reparative and restorative changes in the chronic kidney disease (CKD) nephron. We present our protocol and preliminary analysis of an IRB-approved, phase I single-group, open-label trial that tests the safety and efficacy of Renal Autologous Cell Therapy (REACT; NCT 04115345) in adults with congenital anomalies of the kidney and urinary tract (CAKUT). Methods: Adults with surgically corrected CAKUT and CKD stages 3 and 4 signed an informed consent and served as their “own” baseline control. REACT is an active biological ingredient acquired from a percutaneous tissue acquisition from the patient’s kidney cortex. The specimen undergoes a GMP-compliant manufacturing process that harvests the selected renal cells composed of progenitors for renal repair, followed by image-guided locoregional reinjection into the patient’s renal cortex. Participants receive 2 doses at 6-month intervals. Primary outcomes are stable renal function and stable/improved quality of life. Additional exploratory endpoints include the impact of REACT on blood pressure, vitamin D levels, hemoglobin, hematocrit and kidney volume by MRI analysis. Results: Four men and 1 woman were enrolled and underwent 5 cell injections. Their characteristics were as follows: mean 52.8 years (SD 17.7 years), 1 Hispanic, 4 non-Hispanic, and 5 white. There were no renal tissue acquisition, cell injection, or cell product-related complications at baseline. Conclusion: REACT is demonstrating feasibility and patient safety in preliminary analysis. Autologous cell therapy treatment has the potential to stabilize or improve renal function in CAKUT-associated CKD to delay or avert dialysis. Patient enrollment and follow-up are underway.

MP19-07 CAN WE USE HUMAN ADIPOSE-DERIVED STEM CELLS (ADSCS) FROM UROLOGIC CANCER PATIENTS FOR AUTOLOGOUS CELL THERAPY?: A PILOT STUDY

2015 ◽  
Vol 193 (4S) ◽  
Author(s):  
Marta Garcia-Contreras ◽  
Cesar Vera-Donoso ◽  
José Hernández-Andreu ◽  
José García-Verdugo ◽  
Elisa Oltra
Keyword(s):  
Stem Cells ◽  
Pilot Study ◽  
Cell Therapy ◽  
Cancer Patients ◽  
Adipose Derived Stem Cells ◽  
Autologous Cell ◽  
Urologic Cancer ◽  
Autologous Cell Therapy

Autologous cell therapy for cardiac repair

2011 ◽  
Vol 11 (4) ◽  
pp. 489-508 ◽  
Author(s):  
Darryl R Davis ◽  
Duncan J Stewart
Keyword(s):  
Cell Therapy ◽  
Cardiac Repair ◽  
Autologous Cell ◽  
Autologous Cell Therapy
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