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EP-4735586-A1 - CELLULAR REPROGRAMMING

EP4735586A1EP 4735586 A1EP4735586 A1EP 4735586A1EP-4735586-A1

Abstract

The present invention relates generally to compositions for cellular reprogramming of human somatic cells into induced neural precursor cells, methods of making induced human neural precursor cells by cellular reprogramming and methods of using reprogramed induced human neural precursor cells for treating disease.

Inventors

  • Samuel, Amy Jane
  • CONNOR, BRONWEN JANE

Assignees

  • Samuel, Amy Jane
  • Connor, Bronwen Jane

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. WHAT WE CLAIM: 1. A composition comprising a basal brain medium and at least two active agents selected from the group consisting of Activin A (ActA), a protein kinase C (PKC) inhibitor, a p160ROCK inhibitor and N-2 supplement.
  2. 2. The composition of claim 1 comprising a basal brain medium and at least three active agents selected from the group consisting of Activin A (ActA), a protein kinase C (PKC) inhibitor, a p160ROCK inhibitor and N-2 supplement.
  3. 3. The composition of claim 1 or claim 2 comprising a basal brain medium and all four active agents Activin A (ActA), a protein kinase C (PKC) inhibitor, a p160ROCK inhibitor and N-2 supplement.
  4. 4. The composition of any one of claims 1 to 3 wherein the PKC inhibitor is selected from the group consisting of Gö6983, Enzastaurin, Staurosporine, GF 109203X, Go6976, Ro 31-8220 mesylate, Ro 32-0432 hydrochloride, Sotrastaurin and K252a, preferably wherein the PKC inhibitor is Gö6983.
  5. 5. The composition of any one of claims 1 to 4 wherein the p160ROCK inhibitor is selected from the group consisting of is Y27632, Thiazovivin, HA 1100 hydrochloride and GSK429286A, preferably wherein the p160ROCK inhibitor is Y27632.
  6. 6. A method of making a human induced lateral ganglionic eminence precursor cell (hiLGEP) comprising: a) reprogramming a human fibroblast (HF) into a hiLGEP comprising a. transfecting the HF with SOX2 cmRNA and PAX6 cmRNA, b. culturing the transfected HF in a composition comprising a basal brain medium, and at least two active agents selected from the group consisting of a protein kinase (PKC) inhibitor, a p160ROCK inhibitor, and N-2 supplement, c. passaging the HF in b. into a composition comprising a basal brain medium and at least three active agents selected from the group consisting of a protein kinase (PKC) inhibitor, a p160ROCK inhibitor, N-2 supplement and Activin A (ActA), and d. culturing the passaged HF.
  7. 7. The method of claim 6 wherein the SOX2 cmRNA comprises (SEQ ID NO:1).
  8. 8. The method of claim 6 or claim 7 wherein the PAX6 cmRNA comprises (SEQ ID NO:2).
  9. 9. The method of any one of claims 6 to 8 wherein the composition in b. comprises a basal brain medium and all three active agents that are a protein kinase (PKC) inhibitor, a p160ROCK inhibitor, and N-2 supplement.
  10. 10. The method of any one of claims 6 to 9 wherein the composition in c. comprises a basal brain medium and all four active agents that are a protein kinase (PKC) inhibitor, a p160ROCK inhibitor, N-2 supplement and Activin A (ActA).
  11. 11. The method in any one of claims 6 to 10 wherein the PKC inhibitor in b. and/or in c. is selected from the group consisting of Gö6983, Enzastaurin, Staurosporine, GF 109203X, Go6976, Ro 31- 8220 mesylate, Ro 32-0432 hydrochloride, Sotrastaurin and K252a, preferably wherein the PKC inhibitor is Gö6983.
  12. 12. The method of any one of claims 6 to 11 wherein the p160ROCK inhibitor in b. and/or in c. is selected from the group consisting of is Y27632, Thiazovivin, HA 1100 hydrochloride and GSK429286A, preferably wherein the p160ROCK inhibitor is Y27632.
  13. 13. The method of any one of claims 6 to 12 wherein the HF is a human dermal fibroblast (HDF).
  14. 14. The method of any one of claims 6 to 12 wherein the HF is an adult human fibroblast (aHF), preferably an adult human dermal fibroblast (aHDF).
  15. 15. A kit comprising: i. a composition comprising a basal brain medium, and at least two active agents selected from the group consisting of a protein kinase (PKC) inhibitor, a p160ROCK inhibitor, and N-2 supplement, and ii. a composition comprising a basal brain medium and at least three active agents selected from the group consisting of a protein kinase (PKC) inhibitor, a p160ROCK inhibitor, N-2 supplement and Activin A (ActA).
  16. 16. The kit of claim 15 wherein the composition in i. comprises all three active agents that are a protein kinase (PKC) inhibitor, a p160ROCK inhibitor, and N-2 supplement.
  17. 17. The kit of claim 15 or claim 16 wherein the composition in ii. comprises all four active agents that are a protein kinase (PKC) inhibitor, a p160ROCK inhibitor, N-2 supplement and Activin A (ActA).
  18. 18. The kit in any one of claims 15 to 17 wherein the PKC inhibitor in i. and or ii. is selected from the group consisting of Gö6983, Enzastaurin, Staurosporine, GF 109203X, Go6976, Ro 31-8220 mesylate, Ro 32-0432 hydrochloride, Sotrastaurin and K252a, preferably wherein the PKC inhibitor is Gö6983.
  19. 19. The kit of any one of claims 15 to 18 wherein the p160ROCK inhibitor in i. and/or in ii. is selected from the group consisting of is Y27632, Thiazovivin, HA 1100 hydrochloride and GSK429286A, preferably wherein the p160ROCK inhibitor is Y27632.
  20. 20. A human induced lateral ganglionic eminence precursor cell (hiLGEP).

Description

CELLULAR REPROGRAMMING 1. FIELD OF THE INVENTION The present invention relates generally to compositions for cellular reprogramming of human somatic cells into induced neural precursor cells, methods of making induced human neural precursor cells by cellular reprogramming and methods of using reprogramed induced human neural precursor cells for treating disease. 2. BACKGROUND TO THE INVENTION Huntington’s disease (HD) is a genetic neurological disorder caused by an expansion mutation of the trinucleotide (CAG) repeat in exon 1 of the HTT (IT15) gene, encoding a 350-kDa protein termed Huntingtin (HTT). The disease is inherited in an autosomal dominant manner and shows a prevalence of about 1 to 15,000 individuals. HD is characterised by neuronal cell loss mainly in the caudate nucleus, putamen and the cerebral cortex. In later stages, areas such as the hippocampus and hypothalamus are affected (Vonsattel et al., 1985). Predominant degeneration of medium-sized spiny striatal projection neurons (MSNs) results in motor dysfunction together with cognitive and psychiatric disturbances. Current treatment options for HD are severely limited. While some of the behavioural symptoms of HD respond to psychiatric treatments and several drugs are available to reduce the impact of chorea, other motor symptoms and the cognitive symptoms of HD are currently not treatable (Caron et al., 1998). Cell transplantation is a viable option for the treatment of HD with the aim of reconstructing the damaged neural circuitry by replacing cells lost to the disease process, with the expectation that the donor cells will reconnect to remaining host neural networks to repair connectivity. Through genetic testing early therapeutic intervention is possible allowing for transplantation of replacement MSNs prior to extensive degeneration with the aim of maintaining the corticostriatal circuitry. Both rodent and primate HD studies have demonstrated that reconstruction of the corticostriatal circuitry following cell transplantation can alleviate both motor and cognitive deficits observed in HD (Dunnett et al., 2000; Kendall et al., 1998; Palfi et al., 1998). Small open-label clinical trials investigating the transplantation of human fetal striatal tissue into HD patients have been performed and provide preliminary proof of principle that neural transplantation can benefit patients with HD (Rosser & Bachoud-Levi, 2012). However, for cell transplantation therapy to be a viable therapeutic option for HD patients, one of the main issues that needs to be addressed is the identification of an ethically and technically viable source of donor cells other than human fetal striatal tissue. In searching for an alternative donor cell source attention has fallen on the potential use of human- derived stem cells including human embryonic stem cells (hESCs) or human induced pluripotent stem cells (iPSC) (Connor, 2018). While initial studies demonstrated that hESC-derived neural stem cells (NSC) transplanted into the quinolinic acid (QA) lesion model of HD survive and generate new neurons, transplanted human NSCs did not differentiate into region-specific neurons expressing markers of MSNs (Joannides et al., 2007; Reidling et al., 2018; Song et al., 2007; Vazey et al., 2010). To increase lineage specificity and encourage differentiation into MSNs, several groups differentiated hESCs into striatal precursors (Arber et al., 2015; Aubry et al., 2008; Delli Carri et al., 2013; Faedo et al., 2017; 2013). These studies reported the generation of MSNs following transplantation of striatal precursors into the QA lesioned striatum demonstrating the requirement to drive hESCs to a lineage-specific precursor fate before transplantation. Several studies have also demonstrated the potential use of iPSC-derived NSCs as a cellular source for transplantation therapy for HD (An et al., 2012; Jeon et al., 2012), with An and colleagues (An et al., 2012) demonstrating the capability to transplant genetically corrected HD patient- derived cells. However, the use of hESC- or hiPSC-derived NSCs for cell transplantation comes with the potential risk of tumorigenesis and genetic mutagenesis due to the accumulation of chromosomal abnormalities associated with long-term passaging. Furthermore, hiPSCs carry the risk of developing genetic abnormalities and insertional mutagenic effects due to the oncogenic nature of reprogramming factors and the integrative methods of gene delivery used in the reprogramming process (González et al., 2011). In view of the above, there is a need in the art for the development of alternative forms of cellular transplantation therapy that avoid the problems identified with the use of hESC- or hiPSC-derived NSCs, particularly the risks associated with tumorigenesis, genetic mutagenesis, development of genetic abnormalities and insertional mutagenic effects. It is an object of the invention to provide compositions and methods that will support at least one su