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CN-121986157-A - Cell therapy

CN121986157ACN 121986157 ACN121986157 ACN 121986157ACN-121986157-A

Abstract

A cell population for treating heart disease and a method of making the same.

Inventors

  • K. J. Shetland
  • WANG QINGDONG
  • M. FELLOWS

Assignees

  • 阿斯利康(瑞典)有限公司

Dates

Publication Date
20260505
Application Date
20240916
Priority Date
20230918

Claims (20)

  1. 1. A population of cells, wherein: a) At least 70% of the cells in the population express a Cardiac Progenitor (CPC) -related marker, and B) 1% or less of the cells in the population express octameric transcription factor 4 (OCT 4).
  2. 2. The population of cells of claim 1, comprising, consisting of, or consisting essentially of dissociated cells.
  3. 3. The population of cells of claim 1 or 2, comprising, consisting of, or consisting essentially of dissociated single cells.
  4. 4. The population of cells of any preceding claim comprising, consisting of, or consisting essentially of cells in suspension.
  5. 5. The cell population of any preceding claim, wherein the cell population is committed to the heart lineage, optionally wherein the cell population is committed to the ventricular heart lineage.
  6. 6. The cell population of any preceding claim, wherein less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, or 0.2% of the cells in the cell population express OCT4.
  7. 7. The cell population of any preceding claim, wherein about 0.1% of the cells in the cell population express OCT4.
  8. 8. The cell population of any preceding claim, wherein no cells in the cell population express OCT4.
  9. 9. The cell population of any preceding claim, wherein the percentage (%) of cells in the cell population that express OCT4 is determinable by flow cytometry, single cell RNA sequencing, or immunofluorescence, or by flow cytometry, single cell RNA sequencing, or immunofluorescence.
  10. 10. The population of cells according to any preceding claim, wherein 1.5% or less of the cells in said population express T cell receptor alpha locus 1-60 (TRA-1-60).
  11. 11. The cell population of any preceding claim, wherein less than 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3% or 0.2% of the cells in the cell population express TRA-1-60.
  12. 12. The population of cells of any preceding claim, wherein 1.5% or less of the cells in the population express TRA-1-60.
  13. 13. The cell population of any preceding claim, wherein the percentage (%) of TRA-1-60 expressing cells in the cell population is determinable by or by flow cytometry, single cell RNA sequencing, or immunofluorescence.
  14. 14. The cell population of any preceding claim, wherein OCT4 expression in the cell population is below detection limit as measured by flow cytometry, microarray, RNA sequencing, and/or quantitative Polymerase Chain Reaction (PCR).
  15. 15. The population of cells according to any preceding claim wherein 1% or less of the cells in the population express NANOG.
  16. 16. The cell population of any preceding claim, wherein less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% of the cells in the cell population express NANOG.
  17. 17. The cell population of claim 15 or 16, wherein the percentage (%) of NANOG-expressing cells in the cell population is determinable by flow cytometry, single cell RNA sequencing, or immunofluorescence, or by flow cytometry, single cell RNA sequencing, or immunofluorescence.
  18. 18. The population of cells of any preceding claim, wherein 3% or less of the cells in the population express SOX2.
  19. 19. The cell population of any preceding claim, wherein less than 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3% of cells in the cell population express SOX2.
  20. 20. The cell population of claim 18 or 19, wherein the percentage (%) of SOX2 expressing cells in the cell population is determinable by flow cytometry, single cell RNA sequencing, or immunofluorescence, or by flow cytometry, single cell RNA sequencing, or immunofluorescence.

Description

Cell therapy 1. Background art It is estimated that 6400 tens of thousands of people worldwide suffer from Heart Failure (HF), with a prevalence of HF known to be about 1% to 2% of the total adult population. HF results in over one million hospitalizations annually in the united states and europe [1]. Once diagnosed, HF patients are hospitalized once a year on average [2]. Due to the combination of increased prevalence of population growth, aging and complications, the absolute number of HF hospitalizations is expected to increase in the future, possibly as much as 50% [2] within 25 years. HF with low ejection fraction (LVEF) (i.e., < 40%) accounts for about half of all HF cases [3,4]. Primary drug treatments for HF include beta blockers, angiotensin converting enzyme inhibitors, angiotensin receptor enkephalinase inhibitors, SGLT2 inhibitors, mineralocorticoid receptor antagonists, diuretics for fluid retention and hydralazine/nitrate therapy for selected patients [5]. Although existing cardiac HF care standards include pharmacological treatment with several available classes of drugs, devices and cardiac transplants, all have certain limitations, as the 1-year mortality of advanced HF patients is in the range of 25% to 75%, there is still a significant unmet medical need [2]. Guidelines have recently been updated to incorporate, for example, SGLT2 inhibitors and angiotensin receptor enkephalinase inhibitors, but improving cardiac function and prognosis remains challenging. Many therapies are also in development for HF, but options for patients with advanced HF remain limited. Foo et al [6] describe a method for generating ISL 1-positive human ventricular progenitor cells ("human ventricular progenitor cells", HVP) from human Embryonic Stem Cells (ESCs) capable of differentiating into ventricular myocardium in vivo. The HVP population was harvested on day 6 of differentiation and Foo et al identified this HVP population as the best differentiation window period [6]. WO 2016/029122A 1[7] describes a population of human cardiac ventricular progenitor cells derived from ESCs harvested on day 6 of differentiation and having been contacted with one or more reagents reactive with Jagged 1 (JAG 1) and/or Frizzled 4 (FZD 4). WO 2017/172086 a1[8] describes genetic markers JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9 for identifying implantable human ventricular progenitor cells. HVP was harvested from ESC on day 6 of differentiation. WO 2018/100433 a1[9] describes a method of isolating human cardiac ventricular progenitor harvested on days 5 to 7 of differentiation. WO 2019/038587 a1[10] describes a method for isolating human cardiac ventricular progenitor comprising contacting a human cell culture containing cardiac progenitor with one or more agents having reactivity with neuropilin-1 (NRP 1). 2. Summary of the invention The risk associated with cells derived from pluripotent stem cells is teratoma formation. The present disclosure, supported by the data presented for the first time herein, is based on a new ventricular progenitor differentiation protocol that reduces the risk of teratoma formation and provides a population of cells suitable for use as a cell therapy product for administration to a patient. 3. Description of the drawings FIG. 1 scheme for differentiation of cardiac progenitor cells Differentiation of a cell population of cells suitable for repair of cardiac ventricular tissue includes obtaining a population of pluripotent stem cells in culture. On day 0, cells are cultured in the presence of a GSK inhibitor (e.g., CHIR). On day 1, GSK inhibitors were removed. On day 3, cells were cultured in the presence of WNT inhibitor. On day 5, wnt inhibitors were removed. On day 8, differentiated progenitor cells were harvested and subjected to TRA-1-60 sorting to eliminate TRA-1-60 expressing cells. PSC, pluripotent stem cells, VPC, cardiac ventricular progenitor cells, MACS, magnetically activated cell sorting. Foo et al describe methods for early differentiation of ventricular progenitor cells (harvested on day 6). FIG. 2 Gene expression in cardiac progenitor cells PCA plots of RNAseq data from day 0 (pluripotent cells) and days 5 to 10 (VPC) and day 15 (immature cardiomyocytes). FIG. 3 teratoma formation of cardiac progenitor cell populations implanted into the kidney of mice to day 6 Unsorted day 6 cardiac progenitor cells were injected into the kidney capsule and grown out on the kidneys of mice. Left, macroscopic image of teratoma, right, histological appearance of teratoma (HE) FIG. 4 OCT4 and TRA-1-60 expression by day 6 and day 8 harvested cells Cell populations harvested on days 6 and 8 were subjected to TRA-1-60 sorting (depletion of TRA-1-60 expressing cells). FIG. 5 detection of residual pluripotent cells by qPCR, FACS and HEC assays HEC, high-efficiency culture. FACS, fluorescence activated cell sorting, qPCR, quantitative polymerase chain reaction. FIG. 6 expression of undifferentiated marker in the