Stable RNA molecule prepared in vitro and use thereof in preparation of in-vivo in-situ car treatment product

Abstract

A stable RNA molecule prepared in vitro and a use thereof in preparation of an in-vivo in-situ CAR treatment product, wherein mRNA is redesigned, and protective RNA secondary structures are added to two ends of the mRNA to protect the mRNA from being cut by an RNA exonuclease, thereby improving the stability of RNA; moreover, IRES is used for replacing a 5'-terminal m7G cap structure to recruit ribosomes to start protein translation, so that the mRNA retains a translation function, the stability of the mRNA is improved, and there is no need to add caps and tails to the two ends of the mRNA, thereby greatly reducing the preparation cost of the mRNA, and improving the in-vitro production efficiency of the mRNA. Besides, on this basis, a polycistronic RNA platform is constructed, IRESs are used for expressing a plurality of protein sequences, respectively, making up for the deficiency that monocistronic RNA technology cannot be used to simultaneously express a plurality of functional proteins.

Description

A stable RNA molecule prepared in vitro and its application in the preparation of in situ CAR therapeutic products in vivo Technical Field

[0001] The present invention relates to a stable RNA molecule prepared in vitro and its application in the preparation of an in situ CAR therapeutic product in vivo, belonging to the field of biomedicine technology. Background Art

[0002] The concept of CAR (Chimeric Antigen Receptor) was first proposed by scientist G. Gross in 1989. Since then, CAR-T (Chimeric Antigen Receptor T Cell) therapy, which genetically engineered CAR molecules into T cells in vitro, has become an important immunotherapy and has been approved for clinical use in the treatment of leukemia and lymphoma. However, this therapy still faces many challenges. Its application is cumbersome and expensive, and it often requires combination therapy with other medical treatments to achieve optimal results. Furthermore, it is currently ineffective against solid tumors.

[0003] Polycistronic expression refers to a single mRNA molecule capable of encoding multiple polypeptide chains simultaneously. It is primarily found in prokaryotes and less commonly in eukaryotes. Currently, most widely used vectors utilize a polycistronic expression strategy, utilizing elements such as an internal ribosome entry site (IRES) or self-cleaving 2A peptide (2A) to link multiple genes, achieving expression from a single vector. This technique uses DNA plasmids as vectors, and when introduced into target cells, there is a risk of genomic integration.

[0004] RNA vaccine technology, including mRNA vaccine technology and circular RNA vaccine technology, is a medical technology that has achieved major breakthroughs in recent years. Although this technology has shown broad application prospects in the prevention and treatment of infectious diseases, autoimmune diseases, genetic diseases and cancer, it still has huge room for optimization and improvement. Current RNA technologies are all monocistronic RNA technologies, that is, one RNA molecule can only translate into one polypeptide (a single protein subunit or a single fusion protein). Therefore, in application scenarios where multiple functional proteins need to be expressed simultaneously (such as multivalent vaccines, combined drug administration, etc.), current monocistronic RNA technologies encounter technical application bottlenecks.

[0005] In recent years, immune checkpoint inhibition (ICB) and chimeric antigen receptor T cell (CAR-T) immunotherapy have become important cancer treatments, but many challenges remain. ICB therapy has a very low clinical response rate of only 20%-30%, making it virtually ineffective for most cancer patients. While CAR-T therapy has achieved significant success in treating B lymphoma, it still faces three serious challenges: ① It requires time-consuming, resource-intensive, and extremely expensive personalized cell therapy, rather than an off-the-shelf drug; ② It is only effective against some hematologic malignancies and has little to no effect on solid tumors; and ③ It can be associated with severe side effects, such as cytokine storms and killing of normal cells. Therefore, the development of cutting-edge biotechnologies for cancer treatment remains urgently needed.

[0006] RNA therapy technology has the advantages of being reversible and controllable. RNA can directly translate proteins in the cytoplasm to function without being transported into the nucleus; and RNA does not have the safety risks of integrating into genomic DNA. Therefore, RNA therapy technology is an important opportunity to develop in situ CAR immunotherapy in vivo. In November 2020, Matthias Stephan's team directly intravenously infused mRNA encoding the Anti-CD19 CAR transmembrane protein into mice repeatedly, and the therapeutic effect achieved the efficacy of adoptive cell therapy in the mouse model (Parayath et al., Nature Communications, 2020). In January 2022, JOEL G. RURIK et al. reported an in vivo reprogramming CAR-T therapy based on mRNA technology. By targeted delivery of Anti-FAP-CAR mRNA to T cells in vivo, fibrotic cardiomyocytes in mice were eliminated (Rurik et al., Science, 2022).

[0007] At present, CAR therapy often improves its therapeutic effect by combining it with multiple functional proteins, such as cytokines, monoclonal antibodies, vaccines, small molecule targeted drugs, etc. If the CAR molecule and the above multiple functional proteins can be expressed in vivo simultaneously on the same RNA molecule and act synergistically, it will help improve the targeting and tumor killing effect of CAR therapy and effectively improve the response rate of solid tumor treatment. However, since the cytokines and PDZ domains that promote inflammation and immune cell infiltration are transmembrane or secretory proteins, the use of current monocistronic RNA technology to encode them on the same RNA molecule is subject to technical limitations. The main reasons why current monocistronic RNA technology cannot be used to simultaneously express multiple functional proteins for immunotherapy are: (1) The process of lipid nanoparticles (LNPs) encapsulating RNA molecules is random, and the number of RNA molecules encapsulated by one LNP particle is limited; therefore, when multiple monocistronic RNAs are encapsulated with LNPs, the parallelism, stability and effectiveness of the prepared LNP-RNA drug batches will be severely restricted. (2) Due to the special mechanism of sorting secretory proteins and membrane proteins in mammalian cells (guidance and cleavage of signal peptides), the strategy of using one RNA molecule to translate a fusion protein (each protein subunit is connected by 2A) is only applicable to the combination of cytoplasmic proteins, nuclear proteins or a single transmembrane protein with other cytoplasmic proteins, and cannot be applied to the fusion translation of multiple secretory proteins or transmembrane proteins; however, most functional proteins such as antigens, therapeutic antibodies, cytokines, and chimeric antigen receptors (CARs) used in vaccines are secretory proteins or membrane proteins. For the above reasons, the development of polycistronic RNA technology, that is, the simultaneous translation of multiple functional secretory proteins or membrane proteins using one RNA molecule, is urgently needed in the current field of underlying RNA technology research, and it has great application and development prospects in the treatment of diseases in the future.

[0008] Furthermore, in vivo, mRNA has a short half-life and poor stability. Naked mRNA, when introduced directly into the body, is easily degraded by the ubiquitous RNases. While circRNA is more stable than mRNA, it can easily cause immunogenicity in the body, affecting therapeutic efficacy and limiting its application in medical treatment. Furthermore, when mRNA is prepared in vitro, it requires the addition of a 5' cap structure and a 3' poly A tail. While circRNA does not require capping or tailing, it does require a cyclization reaction. The preparation process is cumbersome, severely limiting production efficiency and increasing costs.

[0009] Summary of the Invention

[0010] In order to solve the above problems, the present invention provides a stable RNA molecule prepared in vitro and its use in the preparation of in situ CAR therapeutic products in vivo.

[0011] The first object of the present invention is to provide a stable RNA molecule prepared in vitro, the RNA molecule comprising:

[0012] The region encoding the protein of interest; and,

[0013] An IRES sequence is provided upstream of the coding region of the target protein; and

[0014] Protective RNA secondary structures are provided at both ends of the RNA molecule.

[0015] Furthermore, the protective RNA secondary structure is a small nucleolar RNA (SnoRNA) sequence and / or a Triplex sequence.

[0016] Furthermore, the small nucleolar RNA sequence is selected from one or more of H / ACA box snoRNAs, C / D box snoRNAs, and small Cazar body RNAs (scaRNAs).

[0017] Furthermore, the Triplex sequence is an RNA sequence with a triple helical structure.

[0018] Furthermore, the RNA molecule further comprises:

[0019] The 5' end is provided with a 5'UTR sequence following the protective RNA secondary structure, and the 3' end is provided with a 3'UTR sequence preceding the protective RNA secondary structure.

[0020] Furthermore, the target protein is one or more.

[0021] Furthermore, the target protein is one or more antibodies; or one or more antigens; or one or more gene editing enzymes; or one or more regulatory factors; or one or more fluorescent proteins.

[0022] Furthermore, the target proteins include: human coagulation factor IX (hFIX), pulmonary surfactant protein B antibody (SP-B), vascular endothelial growth factor A (VEGF-A), methylmalonyl-CoA mutase (hMUT), human erythropoietin (hEPO), cystic fibrosis transmembrane conductance regulator (CFTR), Cas9 endonuclease, Cpf1, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs), anti-HIV antibodies, fbr CD19 and CD22 bispecific antibodies, fbr CD3 and CLDN6 bispecific antibodies, Gaussia luciferase (Glue), Firefly luciferase (Flue), and enhanced green fluorescent protein (eGFP).

[0023] Furthermore, when there are multiple target proteins, the IRES sequences upstream of the coding region of each target protein are different.

[0024] Further, the IRES is derived from the IRES sequence of the following viruses: Taura syndrome virus, triathlon tumor virus, Theiler encephalomyelitis virus, simian virus 40, fire ant virus 1, graminearum aphid virus, reticuloendotheliosis virus, fulman polio virus 1, Kashmir bee virus, human rhinovirus 2, human immunodeficiency virus type 1, Himetobi P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, encephalomyocarditis virus, fruit fly C virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic ringspot virus, classical swine fever virus, simian picornavirus, turnip crinkle virus, coxsackievirus B3 or coxsackievirus A.

[0025] Furthermore, when there are multiple target proteins, the IRES sequences upstream of the coding regions of the multiple target proteins are different sequences.

[0026] Furthermore, the 5'UTR sequence is selected from the RNA binding protein family or the YTHDF family; the 3'UTR sequence is selected from human α-globin 1 (HBA1), human α-globin 2 (HBA2), mouse α-globin, human β-globin, xrRNA or Apt-eIF4G.

[0027] A second object of the present invention is to provide a method for preparing the stable RNA molecule prepared in vitro, comprising the following steps:

[0028] S1. Amplify the RNA polymerase promoter, protective RNA secondary structure sequence, 5'UTR sequence, 3'UTR sequence, IRES sequence and target protein coding gene sequence respectively, and connect them to the empty vector to obtain the expression vector;

[0029] S2. The expression vector obtained in step S1 is linearized by enzyme digestion, and the RNA molecule is synthesized by in vitro transcription.

[0030] Furthermore, the method further comprises encapsulating the RNA molecule using a delivery system.

[0031] Furthermore, the delivery system includes one or more of lipid nanoparticles, liposomes, lipid complexes, polymer materials, micelles, polypeptides, and protamine.

[0032] The third object of the present invention is to provide the application of the stable RNA molecules prepared in vitro, which includes the application in the preparation of vaccine products or the preparation of in situ CAR therapeutic products in vivo.

[0033] The polycisRNA of the present invention effectively solves the following problems:

[0034] (1) The current problem of individualized adoptive cell therapy being time-consuming, resource-consuming, and extremely costly due to immune rejection of allogeneic transplantation: Based on the polycistronic RNA technology of the present invention, a new "off-the-shelf" immunotherapy of isCAR-T / isCAR-M can be developed. Using polycistronic RNA as a carrier, CAR-immune cells can be directly generated in situ in vivo, and the efficacy can be enhanced through the synergistic action of multiple effector proteins. Due to the characteristics of mRNA, the developed immunotherapy avoids the complex process of individualized treatment, has dose-time-dependent efficacy characteristics, and is very clinically safe.

[0035] (2) The present invention modifies the mRNA structure, removes the 5' cap structure and 3' poly A tail while retaining the mRNA protein translation function, and relies on the SnoRNA sequence or Triplex sequence to improve the mRNA stability. During the production process, the product can be obtained by only in vitro transcription using DNA as a template, without the need for capping, tailing or cyclization steps, which greatly saves production time and reduces production costs.

[0036] (3) The present invention uses only one mRNA molecule to express multiple effector proteins, which is different from the multiple mRNA molecules required to express different proteins with monocistronic RNA. This avoids the problem of drug component homogeneity during the drug-making process, improves safety and drug batch parallelism, and provides a guarantee for later clinical applications.

[0037] The beneficial effects of the present invention are:

[0038] The present invention redesigns mRNA, adding protective RNA secondary structures at both ends of the mRNA to protect the mRNA from being cleaved by RNA exonucleases, improving the stability of the RNA, and using IRES to replace the 5'-end m7G cap structure to recruit ribosomes to initiate protein translation, so that the mRNA retains its translation function and increases its stability, without the need for capping and tailing at both ends. This greatly reduces the cost of mRNA preparation and improves the efficiency of mRNA in vitro production. In addition, on this basis, the present invention constructs a polycistronic RNA platform, using IRES to express multiple protein sequences separately, making up for the deficiency of single cistronic RNA technology that cannot express multiple functional proteins simultaneously. Description of the drawings:

[0039] FIG1 is a flow cytometric analysis result 72 hours after transfection of linear RNA encoding green fluorescent protein into 293T cells;

[0040] FIG2 is a graph showing the number of green fluorescent protein-positive cells analyzed by flow cytometry 48 hours after 293T cells were transfected with the linear RNA obtained after IVT of the plasmid constructed in Example 1;

[0041] FIG3 is a histogram analysis of the analysis results of FIG2 ;

[0042] FIG4 is an analysis diagram of the MFI results of the flow cytometry analysis in FIG2 ;

[0043] FIG5 is a conceptual diagram of CAR therapy according to Example 3. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0045] Abbreviations:

[0046] GFP Green fluorescent protein Green fluorescent protein

[0047] ORF Open reading frame

[0048] IRES Internal ribosome entry site Internal ribosome entry site

[0049] UTR Untranslated region

[0050] HEK Human embryonic kidney

[0051] EMCV Encephalomyocarditis virus

[0052] Table 1 Commonly used protective RNA secondary structure sequences

[0053] Table 2 Commonly used IRES sequences

[0054] Table 3 Other IRES sequences

[0055] Table 4 5'UTR sequence

[0056] Table 5 3'UTR sequences

[0057] Example 1:

[0058] 1) Vector construction

[0059] PCR amplification of the T7 promoter, SnoRNA116-14 sequence, 5'UTR sequence, CVB3IRES sequence, sequence encoding green fluorescent protein, 3'UTR sequence, and SnoRNA116-13 sequence. The CVB3IRES sequence and sequence encoding green fluorescent protein were sequentially connected to the vector backbone. Randomly generated sequences were then added at both ends to generate Random1-Random2 linear RNA. SnoRNA116-14 and snoRNA116-13 sequences were added at both ends to generate snoRNA1-snoRNA2 linear RNA. A 5'UTR sequence was added before IRES, and a 3'UTR sequence was added after the sequence encoding green fluorescent protein to form a backbone vector. SnoRNA and / or Triplex sequence and / or random sequence were added to the protection structure sites at both ends to form snoRNA1-snoRNA2(U)Random1-snoRNA2(U), Random3-snoRNA(U), Triplex-snoRNA2(U)snoRNA1-Random2(U), and snoRNA1-Triplex110(U) linear RNAs.

[0060] The vector constructed according to the present invention can be digested with the restriction endonuclease PmeI to convert the circular plasmid into a linear plasmid. The in vitro transcription (IVT) reaction using T7 RNA polymerase eliminates the need for additional capping and tailing reactions during mRNA preparation, nor does it require the series of circularization and delinearization steps required after the circular RNA in vitro transcription (IVT) reaction, thereby generating stable linear RNA in a single step.

[0061] 2) Preparation of delivery system

[0062] Purified linear RNA was encapsulated in liposomes (lipo2000). Linear RNA and lipo2000 were mixed at a ratio of 1:1.5 and transfected into 293T cells. Flow cytometry analysis was performed 72 hours after transfection. The experimental results are shown in Figures 2-4. These results demonstrate that the linear RNA molecules constructed in this invention can stably express green fluorescent protein in cells and have room for optimization. Specifically, linear RNA molecules with sno116-14 at the 5' end and a triplex at the 3' end showed the best expression. Linear RNA molecules with random sequences at both ends showed poor expression.

[0063] Example 2: Infectious Disease Vaccines

[0064] This example takes the COVID-19 vaccine as an example:

[0065] 1) Vector construction

[0066] The T7 promoter, snoRNA and / or triplex sequences, 5'UTR sequence, IRES1, IRES2, IRES3, 3'UTR sequence, snoRNA and / or triplex sequences were amplified by PCR and ligated into a vector containing a selection marker via Gibson Assmble to construct the polycisRNA-EV empty vector. Next, the RBD antigen coding sequences for different SARS-CoV subtypes were amplified by PCR and cloned into the polycisRNA-EV backbone to construct the corresponding polycisRNA plasmids for the subsequent IVT reaction.

[0067] The vector constructed according to the present invention can be digested with the restriction endonuclease PmeI to convert the circular plasmid into a linear plasmid. T7 RNA polymerase is then used for in vitro transcription (IVT) without the need for additional capping and tailing reactions during mRNA preparation, or the subsequent circularization and delinearization steps required for circular RNA in vitro transcription (IVT), resulting in a single-step production of stable linear polycisRNA.

[0068] 2) Preparation of delivery system

[0069] Purified polycisRNA is encapsulated within lipid-based nanoparticles (LNPs). The resulting polycisRNA / delivery nanoparticle assembly undergoes concentration, buffer exchange, and sterile filtration. It is then evaluated and formulated into a vaccine that can be directly administered to humans to prevent disease.

[0070] Example 3: Cancer Vaccine

[0071] This embodiment takes lung cancer as an example:

[0072] 1) Vector construction

[0073] The T7 promoter, snoRNA and / or Triplex sequence, 5'UTR sequence, IRES1 sequence, IRES2 sequence, IRES3 sequence, 3'UTR sequence, snoRNA and / or Triplex sequence were PCR amplified and ligated to a vector containing a selection marker via Gibson Assmble to construct a polycisRNA-EV empty vector. Subsequently, the coding sequences for lung cancer cell surface antigen peptides, programmed death receptor 1 (PD-1) antibody, and epidermal growth factor receptor (EGFR) antibody were PCR amplified and cloned downstream of the IRES1, IRES2, and IRES3 sequences, respectively, to construct the corresponding polycisRNA plasmid for the subsequent IVT reaction.

[0074] The polycisRNA plasmid is digested with the restriction endonuclease PmeI to convert it from a circular plasmid to a linear plasmid. T7 RNA polymerase is then used for in vitro transcription (IVT) to generate a stable linear polycisRNA in one step.

[0075] 2) Preparation of delivery system

[0076] PolycisRNA can be delivered using commonly used delivery systems including lipid nanoparticles (LNP), liposomes, lipid complexes, polymer materials, micelles, polypeptides, protamine, electroporation, etc. This embodiment uses lipid nanoparticles (LNP) to encapsulate the purified polycisRNA therein. The resulting polycisRNA / delivery nanoparticle assembly is concentrated, buffer-exchanged, and sterile-filtered. It is then finally evaluated and packaged in this order to prepare a vaccine, which is then injected into cancer patients to treat the disease.

[0077] Example 4: CAR therapy

[0078] 1) PCR amplify the T7 promoter, snoRNA or / and triplex sequence, 5'UTR sequence, IRES1 sequence, IRES2 sequence, IRES3 sequence, 3'UTR sequence, snoRNA or / and triplex sequence, and connect them to a vector containing a selection marker via Gibson Assmble to construct a polycisRNA-EV empty vector. Next, PCR amplify the chimeric antigen receptor (CAR), cytokine (IL2, etc.), and programmed death receptor 1 (PD-1) antibody coding sequence, clone it into the polycisRNA-EV backbone, and sequentially clone it downstream of the IRES1 sequence, IRES2 sequence, and IRES3 sequence to construct the corresponding polycisRNA plasmid for the subsequent IVT reaction.

[0079] The polycisRNA plasmid is digested with the restriction endonuclease PmeI to convert it from a circular plasmid to a linear plasmid. T7 RNA polymerase is then used for in vitro transcription (IVT) to generate a stable linear polycisRNA in one step.

[0080] 2) Preparation of delivery system

[0081] Lipid nanoparticles (LNPs) that can target T cells are used as a delivery system to encapsulate purified polycisRNA. A certain dose of LNP-polycisRNA is injected into the body as needed for corresponding treatment.

[0082] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A stable RNA molecule prepared in vitro, characterized in that The RNA molecule comprises: The region encoding the protein of interest; and, An IRES sequence disposed upstream of the coding region of the target protein; and Protective RNA secondary structures are provided at both ends of the RNA molecule.

2. The RNA molecule according to claim 1, characterized in that The protective RNA secondary structure is a small nucleolar RNA sequence and / or a Triplex sequence.

3. The RNA molecule according to claim 1, characterized in that The RNA molecule further comprises: The 5'UTR sequence is disposed after the protective RNA secondary structure at the 5' end, and the 3'UTR sequence is disposed before the protective RNA secondary structure at the 3' end.

4. The RNA molecule according to claim 1, characterized in that The target protein is one or more.

5. The RNA molecule according to claim 1, characterized in that The target protein is one or more antibodies; or, one or more antigens; or, one or more gene editing enzymes; or, one or more regulatory factors; or, one or more fluorescent proteins.

6. The RNA molecule according to claim 2, characterized in that The small nucleolar RNA sequence is selected from one or more of H / ACA box snoRNAs, C / D box snoRNAs, and small Cazar body RNAs.

7. The RNA molecule according to claim 1, characterized in that The IRES is derived from the IRES sequence of the following viruses: Taura syndrome virus, triathlon tumor virus, Theiler encephalomyelitis virus, simian virus 40, red fire ant virus 1, graminearum aphid virus, reticuloendotheliosis virus, Fuman polio virus 1, Kashmir bee virus, human rhinovirus 2, human immunodeficiency virus type 1, Himetobi P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, encephalomyocarditis virus, fruit fly C virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic ringspot virus, classical swine fever virus, simian picornavirus, turnip crinkle virus, coxsackievirus B3 or coxsackievirus A.

8. The RNA molecule according to claim 3, characterized in that The 5'UTR is selected from the RNA binding protein family or the YTHDF family; the 3'UTR is selected from human α-globin 1, human α-globin 2, mouse α-globin, human β-globin, xrRNA or Apt-eIF4G.

9. A method for preparing a stable RNA molecule prepared in vitro according to any one of claims 1 to 8, characterized in that: The steps include: S1, respectively amplify RNA polymerase promoter, protective RNA secondary structure sequence, 5'UTR sequence, 3'UTR The sequence, IRES sequence and the coding gene sequence of the target protein are connected to the empty vector to obtain an expression vector; S2. The expression vector obtained in step S1 is linearized by enzyme digestion, and the RNA molecule is synthesized by in vitro transcription.

10. The method according to claim 9, characterized in that The method further comprises encapsulating the RNA molecule using a delivery system.

11. Use of the in vitro prepared stable RNA molecule according to any one of claims 1 to 8, characterized in that: The applications include application in the preparation of vaccine products or in the preparation of in situ CAR therapeutic products.

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