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KR-20260064749-A - Nanoparticles containing a nucleic acid sequence encoding cyclic GMP-AMP synthase

KR20260064749AKR 20260064749 AKR20260064749 AKR 20260064749AKR-20260064749-A

Abstract

The present invention relates to a nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS), a nanoparticle vector containing the same, and uses thereof.

Inventors

  • 마넬 니꼴라
  • 파르디 노르베르트

Assignees

  • 엥스띠뛰 퀴리
  • 더 트러스티스 오브 더 유니버시티 오브 펜실베니아
  • 인스티튜트 내셔널 드 라 싼테 에 드 라 리셰르셰 메디칼르 (인쎄름)

Dates

Publication Date
20260507
Application Date
20240801
Priority Date
20230801

Claims (20)

  1. A nucleic acid comprising a sequence encoding a nuclear localization signal (NLS) and a sequence encoding cyclic GMP-AMP synthase (cGAS) described as SEQ ID NO: 40 or a constantially active variant thereof having at least about 85% identity with it.
  2. In claim 1, the constantially active variant of SEQ ID NO: 40 has a mutation at position 241, said mutation being a nucleic acid that is a substitution, deletion, or insertion.
  3. In claim 1 or 2, the constantially active variant of SEQ ID NO: 40 is a mouse cGAS variant consisting of SEQ ID NO: 1, wherein SEQ ID NO: 1 is a nucleic acid having the R241E mutation.
  4. A nucleic acid according to claim 1 or 2, wherein the permanently active variant of SEQ ID NO: 40 is a mouse cGAS variant consisting of any one of SEQ ID NO: 4, 41, 42, 43, 44, 45, 46, 47 or 48, and SEQ ID NO: 41 has at least one non-R amino acid at position 255 or has no amino acid, or any one of SEQ ID NO: 42-48 has at least one non-R amino acid at position 253 or has no amino acid.
  5. A nucleic acid according to claim 1 or 2, wherein the consistently active variant of SEQ ID NO: 40 is a rat cGAS variant consisting of SEQ ID NO: 49 or SEQ ID NO: 50, wherein SEQ ID NO: 49 has at least one non-R amino acid at position 221 or has no amino acid, or SEQ ID NO: 50 has at least one non-R amino acid at position 233 or has no amino acid.
  6. In paragraph 5, the consistently active variant is the nucleic acid with SEQ ID NO: 51 or 52.
  7. A nucleic acid according to claim 1 or 2, wherein the consistently active variant of SEQ ID NO: 40 is a rat cGAS variant consisting of SEQ ID NO: 53 or 54, wherein SEQ ID NO: 53 has at least one non-R amino acid at position 244 or has no amino acid, or SEQ ID NO: 54 has at least one non-R amino acid at position 256 or has no amino acid.
  8. In paragraph 7, the consistently active variant is the nucleic acid with SEQ ID NO: 55 or 56.
  9. A nucleic acid according to any one of claims 1 to 8, wherein the NLS sequence is a classical nuclear localization signal (cNLS) or a non-classical nuclear localization signal (ncNLS), preferably the NLS sequence is described as any one of SEQ ID NO: 9, 13 to 20 or 39.
  10. In any one of paragraphs 1 to 9, the nucleic acid is a nucleic acid that is mRNA.
  11. In claim 10, the mRNA is a nucleic acid comprising a lateral region, a 5'-cap structure, a chain-terminating nucleotide, a stem loop, a 3'-poly-A tail sequence and/or a polyadenylation signal, preferably a 5'-cap structure and a poly-A tail sequence.
  12. In claim 10 or 11, the nucleic acid is a nucleic acid comprising the sequence as described in SEQ ID NO: 5.
  13. A vector comprising a nucleic acid according to any one of claims 1 to 12, wherein the vector is a nanoparticle, e.g., a lipid-based nanoparticle, a viral nanoparticle, or a dendrimer nanoparticle.
  14. In paragraph 13, the vector is a lipid-based nanoparticle vector comprising a nucleic acid according to any one of paragraphs 1 to 12.
  15. In claim 14, the lipid-based nanoparticle comprises a lipid mixture of ionizable cationic lipids, helper lipids, sterols, and polyethylene glycol-lipids, preferably an ionizable cationic lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and a lipid mixture of polyethylene glycol-lipids.
  16. In paragraph 13, the vector is a viral nanoparticle comprising a nucleic acid according to any one of paragraphs 1 to 12.
  17. In paragraph 13, the vector is a vector that is a dendrimer nanoparticle containing a nucleic acid according to any one of paragraphs 1 to 12.
  18. A pharmaceutical composition comprising a nucleic acid of any one of claims 1 to 12, lipid-based nanoparticles of claim 14 or 15, virus nanoparticles of claim 16, or dendrimer nanoparticles of claim 17, and a pharmaceutically acceptable carrier, or a) a nucleic acid of any one of claims 1 to 12, lipid-based nanoparticles of claim 14 or 15, virus nanoparticles of claim 16 or dendrimer nanoparticles of claim 17, and b) a combination of a separate therapeutic agent, preferably an anticancer agent or an antiviral agent.
  19. In paragraph 18, a pharmaceutical composition or combination product formulated to be suitable for subcutaneous, intramuscular, intratumoral, or intravenous injection, preferably intravenous injection.
  20. A nucleic acid according to any one of claims 1 to 12, lipid-based nanoparticles according to claim 14 or 15, virus nanoparticles according to claim 16, dendrimer nanoparticles according to claim 17, or a pharmaceutical composition according to claim 18 or 19 for use as a drug.

Description

Nanoparticles containing a nucleic acid sequence encoding cyclic GMP-AMP synthase The present invention belongs to the field of medicine. The present invention relates to a nucleic acid(s) or a nanoparticle vector (e.g., a lipid-based nanoparticle) comprising said nucleic acid(s) or said nucleic acid molecule(s), and the use thereof for treating pathological conditions such as cancer and infection. Cellular recognition of exogenous DNA is an evolutionarily conserved mechanism by which the innate immune system detects pathogens in mammals. Circular GMP-AMP synthase (cGAS) and its downstream effector, interferon gene stimulator (STING), are involved in mediating basic innate antimicrobial immunity by promoting the release of type I interferon (IFN) and other inflammatory cytokines. STING signaling has also been identified as a therapeutic target in autoinflammatory disorders and neurological diseases, and its role in neuroinflammation is increasingly recognized. The cGAS-STING pathway is currently being studied as being implicated in the progression of neuroinflammation in hosts with central nervous system (CNS) conditions, including Alzheimer's disease, traumatic brain injury, and amyotrophic lateral sclerosis (ALS) (Fryer et al. , Front. Neurosci., 2021 Volume 15). Finally, activation of the cGAS-STING axis is also important in anti-tumor immunity. Downstream cytokines regulated by cGAS-STING, particularly type I IFN, act as a bridge linking innate immunity to adaptive immunity. Therefore, an increasing number of studies are focusing on the synthesis and screening of STING pathway agonists. To date, various types of STING agonists have been discovered, which are mainly classified into cyclic dinucleotides and their derivatives, the flavone-8-acetic acid derivative 5,6-dimethylxanthenon-4-acetic acid (DMXAA) and its analogs, and small molecule agonists (Zheng et al., Mol Cancer. 2020 Aug 27;19(1):133). However, as emerging evidence suggests a pro-tumor role of the cGAS-STING pathway from tumor initiation and development to metastasis (Jiang et al., J Hematol Oncol 13, 81 (2020)), the application of STING agonists in clinical practice remains a major challenge. The efficacy of STING pathway agonists is also limited due to numerous drug delivery and pharmacological issues. Depending on the class of STING agonists and the preferred route of administration, these may include poor drug stability, immunocytotoxicity, immune-related adverse events, limited tumor or lymph node targeting and/or retention, low cellular uptake and intracellular delivery, and complex dependence on the intensity and kinetics of STING signaling (Garland et al. , Chemical Reviews 2022 122 (6), 5977-6039). Therefore, there is a need to develop improved agents targeting the cGAS-STING axis for safe and effective therapies, particularly for cancer, neurological diseases, and infectious diseases. Summary of the Invention To this end, the inventors developed a nucleic acid molecule comprising a nuclear localization signal (NLS) sequence and a sequence encoding a specific cyclic GMP-AMP synthase (cGAS) as a vector nanoparticle, particularly suitable for intravenous, intramuscular, intratumoral, or subcutaneous administration in patients, as a lipid-based nanoparticle. Vaccines based on mRNA-containing lipid nanoparticles (LNPs) are a promising novel delivery platform. LNPs can be used to deliver mRNA to cells and induce the expression of the encoded protein, thereby providing immune protection to the body. Other types of vector nanoparticles, such as viral nanoparticles or dendrimer nanoparticles, may also be used. In particular, the inventors demonstrate that subcutaneous, intramuscular, intratumoral, or intravenous administration of a vector such as an LNP containing a nucleic acid molecule encoding NLS-cGAS in this specification causes reduced tumor growth, and furthermore, intravenous administration of the LNP causes tumor eradication. Surprisingly, NLS-cGAS derived from rodents such as mice ( Mus musculus or “house rat”), rats (e.g., Rattus Norvegicus , also known as “brown rat”), or Arvicanthis niloticus , also known as “Nile rat”) strongly induces cGAMP production, whereas NLS-cGAS derived from Homo sapiens or macaques ( Macaca mulatta ) has no significant effect. Accordingly, the present invention relates to a nucleic acid comprising a sequence encoding a nuclear localization signal (NLS) described as SEQ ID NO: 1 and a sequence encoding a circular GMP-AMP synthase (cGAS), or a variant thereof having at least about 80% or about 85% identity with the same and an R241 mutation, such as, for example, R241E, R241D, R241N, or R241A substitution. In this specification, the inventors show that the nucleic acid of the present invention, comprising a sequence encoding a nuclear localization signal (NLS) and a cyclic GMP-AMP synthase (cGAS), may be the sequence described as SEQ ID NO: 40, or a variant thereof having at least about 80% or about 85%