Project Portugal 2030

Summary

RETORNA project has 4 specific aims pursued in 4 WPs: Aim 1: NP development and characterization (WP3). The main goal of this WP is to prepare polymeric NPs for the delivery of mRNA encoding dCas9-VPR to astrocytes and to characterize the formulation regarding size, charge, stability to storage and their capacity to load and release the mRNA. Our preliminary results (published) indicate the preparation of polymeric NPs able to complex very efficiently mRNA. The hit NP formulations were identified by high-throughput screening of more than 150 NP formulations using a reporter fibroblast model sensitive to mRNA encoding Cre recombinase (28). In addition, unpublished results showed that the hit NP formulation complexes efficiently dCas9-VPR mRNA together with sgRNA targeting Ngn2, one of the transcription factors that will be used in reprogramming astrocytes into neurons. At the end of this WP, we expect to have a mRNA formulation that is (i) optimized in terms of mRNA:polymer ratio, (ii) conjugated with an antibody to target ACSA-2-positive cells and (ii) stable for long-term storage (~2 weeks) in PBS or in a dried format at temperatures compatible with cold supply chain (higher than -20ºC). Aim 2: In vitro cell reprogramming by a non-viral formulation (WP4). The main goal of this WP is to demonstrate, both in cell models as well as brain organotypic cultures, the cellular reprogramming of astrocytes into neurons mediated by the NP formulation containing mRNA encoding CRISPR/dCas system targeting Ngn2 or Ascl1. Our preliminary results (unpublished) show that our NP formulations are able to transfect mouse astrocytes and induce the expression of Ngn2 or Ascl1. In this WP, we will: (i) evaluate transdifferentiation of astrocytes into neurons by qRT-PCR, cell morphology and immunocytochemistry; (ii) evaluate NP targeting to astrocytes in mouse organotypic cortical slice cultures and (iii) investigate astrocyte reprogramming in organotypic slice cultures. At the end of this WP, we expect to (i) demonstrate the reprogramming of astrocytes into neurons with a success rate above 30% and (ii) demonstrate that astrocytes are the cells preferentially targeted in the organotypic culture. Aim 3: In vivo cell reprogramming by a non-viral formulation (WP5). The main goal of this WP is to demonstrate in vivo the cell reprogramming of astrocytes into neurons mediated by the NP formulation containing mRNA encoding CRISPR/dCas system targeting Ngn2 or Ascl1. Our recent published results showed that the administration of a Cas9 protein NP formulation in the brain of a transgenic reporter Ai9 mice by intracerebral administration (in the ventricles) can gene edit the cells lining the ventricles (Simões et al., Angew Chem Int Ed 2024, accepted). In a separate study, we have shown that the feasibility to inject nanoformulations by intracerebral administration in the lesion area affecting the cortex after a middle cerebral artery occlusion (27). At the end of this WP, we expect to: (i) demonstrate cellular reprogramming of brain cells in vivo after the administration of mRNA-NPs and (ii) investigate the effect of reprogramming in terms of neuroinflammation, angiogenesis, cell death, and microglial activation. Aim 4: Human brain biopsies reprogramming by a non-viral formulation (WP6). The main goal of this WP is to demonstrate in human brain biopsies the cell reprogramming of astrocytes into neurons, reinforcing the clinical relevance of the NP formulation.