83 results about "SaRNA" patented technology

The invention discloses a self-replicating RNA (Ribonucleic Acid) molecule, a porcine rotavirus RNA vaccine and application thereof, and relates to the technical field of vaccines. According to the present invention, the coding sequence of the porcine rotavirus G9P23 type VP4 protein is optimized, such that the fact that the saRNA prepared by using the nucleotide sequence represented by the SEQ ID NO: 10 can secrete the high porcine rotavirus G9P23 type VP4 protein antigen after the cell transfection is found; after the optimized porcine rotavirus G9P23 type VP4 protein is used for immunizing experimental animals, the level of neutralizing antibodies generated by the animals can be improved, and the challenge protection effect is enhanced. Therefore, the nucleic acid molecule provided by the invention can be used for developing drugs and vaccines for causing protective immune response in experimental animal bodies. Compared with a traditional vaccine, the vaccine provided by the invention has good immunogenicity and safety, and has a good application prospect.

The invention discloses an SaRNA vaccine for echinococcosis as well as a preparation method and application of the SaRNA vaccine. The preparation method of the SaRNA vaccine comprises the following steps: carrying out codon optimization on a modified target antigen protein through a genetic engineering technology, then assembling the modified target antigen protein with a self-replicating protein sequence, 5 'UTR, 3' UTR and Poly (A) tail, carrying out gene synthesis, then cloning the synthesized gene into a plasmid, and carrying out purification to obtain the SaRNA vaccine. The preparation method comprises the following steps: constructing recombinant plasmids, sequentially carrying out plasmid linearization, in-vitro transcription and purification on the constructed recombinant plasmids to prepare SaRNA molecules, and finally wrapping the SaRNA molecules in lipid nanoparticles to form the SaRNA vaccine for the echinococcosis. Experiments prove that the SaRNA vaccine can activate humoral immunity and cellular immunity of mice at the same time, high-level EG95 specific antibodies and cytokines can be generated through low-dose immunity, and the SaRNA vaccine has wide application prospects in the aspect of preventing and / or treating the echinococcosis.

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. The RNA polymerase variant provided by the invention has the performance of remarkably improving the integrity of a super-long fragment mRNA transcription product, can be used for preparing mRNA through in-vitro transcription, particularly can improve the integrity of the product when preparing saRNA exceeding 10000 nt, and has important significance for industrial production of saRNA.

The invention relates to RNA constructs encoding (i) at least one therapeutic biomolecule; and (ii) at least one innate inhibitor protein (IIP). The constructs are RNA replicons and saRNA molecules, and the invention includes genetic constructs or vectors encoding such RNA replicons. The invention extends to the use of such RNA constructs and replicons in therapy, for example in treating diseases and / or in vaccine delivery. The invention extends to pharmaceutical compositions comprising such RNA constructs, and methods and uses thereof.

The disclosure relates to saRNAs useful in upregulating the expression of a target gene and therapeutic compositions comprising the saRNAs, wherein the saRNAs are chemically modified. Methods of using the saRNAs and the therapeutic compositions are also provided.

A lipid nanoparticle composition for use in the delivery of a self-amplifying RNA (saRNA) construct in one or more target cells is disclosed. The lipid nanoparticle composition comprises a lipid mixture comprising at least one (ionizable) cationic lipid, at least one helper lipid, a sterol, a corticosteroid such as Dexamethasone, and a least one lipid-polyethylene glycol conjugate. The saRNA construct comprises a first open reading frame which encodes one or more non-structural proteins, and a second open reading frame operatively linked to the first open reading frame. The second open reading frame comprises a coding region which encodes one or more target proteins. In some embodiments, the target proteins comprise one or more therapeutic proteins. In some embodiments, the target proteins comprise one or more one or more Cas proteins and / or variants thereof for use in CRISPR-based gene editing.

The present invention relates to a composition comprising a self-amplifying RNA (saRNA) encoding at least one polypeptide of infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1, GaHV-1), a polypeptide variant of ILTV, or an immunogenic fragment or epitope thereof, and a pharmaceutically acceptable excipient comprising lipid inorganic nanoparticles (LION).More specifically, the sRNA encodes at least one laryngotracheitis virus polypeptide, an ILTV polypeptide variant, or an immunogenic fragment or epitope thereof, said sRNA comprising a first nucleic acid sequence including nsP1, nsP2, nsP3 and nsP4 which encode non-structural alphavirus proteins, a second nucleic acid sequence encoding at least one ILTV polypeptide, an ILTV polypeptide variant, an immunogenic fragment or an epitope thereof, and also comprising a 5' cap, a 5' UTR upstream of the first nucleic acid sequence, a 26S promoter upstream of the second nucleic acid sequence, a 3' UTR and a poly A tail.

The invention provides a phage T7RNA (Ribonucleic Acid) polymerase (T7RNAP) mutant with high performance. The phage T7RNA polymerase (T7RNAP) mutant has mutation at a 744th amino acid residue corresponding to a wild type T7RNA polymerase; further, the T7RNAP mutant comprises an amino acid residue mutation occurring at at least one site selected from the following groups: a 62 site, a 68 site, a 72 site, a 152 site, a 173 site, a 535 site, a 538 site, a 539 site, a 633 site, a 723 site, a 767 site, a 881 site and a 883 site. When the T7RNAP mutant is applied to an in-vitro transcription (IVT) reaction, the yield of a target product in the IVT reaction can be remarkably increased, and / or the formation of a double-stranded RNA (dsRNA) byproduct in the IVT reaction can be reduced, so that the T7RNAP mutant can provide selection and adaptive industrial application for the IVT reaction of mRNA, CircRNA or saRNA and other drug molecules.

The application discloses a three-target-point serial saRNA sequence for activating an NKCC1 gene, an adenovirus vector, a construction method and application, and comprises serial saNKCC1-1, saNKCC1-2 and saNKCC1-3 target site sequences in sequence; the target site sequences are as follows: saNKCC1-1: 5'-TCTGCAAATCCAGGCTCTTTA-3'; saNKCC1-2: 5'-TGCGAAATGGGAAACTAGTAA-3'; and saNKCC1-3: 5'-GCTGGAATTACTCTTTGGTTA-3'; the saNKCC1-1, saNKCC1-2 and saNKCC1-3 are serially connected through linker sequences to form a complete three-target-point serial saRNA sequence. The application can efficiently target and regulate the expression of the NKCC1 gene.

The invention discloses a preparation method and application of a tumor microenvironment response type cascade targeting small activation nucleic acid nano-drug, and belongs to the technical field of biological medicines. According to the present invention, M2 type TAM targeting peptide CRVLRSGSC modified dendrimer PAMAM is adopted to jointly load small activation RNA (sa-p38, sa-TFEB) of p38 and TFEB, and acid-sensitive liposome is adopted to wrap and protect the compound (CRV-PAMAM / saRNA) to prepare the tumor microenvironment response type cascade targeting small activation nucleic acid nano-drug, and the phenotype and the metabolic mode of M2 type TAM can be reprogrammed into M1 type TAM so as to kill tumor cells, such that the tumor microenvironment response type cascade targeting small activation nucleic acid nano-drug can be used for preparing the tumor microenvironment response type cascade targeting small activation nucleic acid nano-drug, and the tumor microenvironment response type cascade targeting small activation nucleic acid nano-drug can be used for preparing the tumor microenvironment response type cascade targeting small activation nucleic acid nano-drug. And a tumor immune microenvironment is remodeled, and finally, tumor growth is inhibited and even tumors are ablated. The invention has a very wide application prospect in gene therapy and immunotherapy of cancers, hereditary diseases, infectious diseases and the like.

The invention discloses an industrial purification method for improving the integrity of self-replicating RNA (Ribonucleic Acid), which comprises the following steps: (1) combining: mixing saRNA subjected to capping with a high-salt ion concentration buffer solution, and loading a sample to a affinity chromatographic column; (2) washing: washing the affinity chromatography column with a low-salt ion concentration buffer solution to remove impurities; and (3) eluting: eluting with DEPC (diethylpyrocarbonate) water, and collecting the eluent in the range of 150 mAU to 150 mAU, so as to obtain the saRNA product. According to the method, the purification process of the saRNA can be rapidly completed, the technological operation process is simple, convenient and rapid, the integrity of the saRNA is obviously improved by 8%-11.4%, the experimental process is easy to amplify, and the method has very strong technical advantages for the technological process for operating the long-fragment saRNA.

The present invention relates to compositions comprising RNA, preferably messenger RNA (mRNA), more preferably self-amplifying RNA (saRNA), and polymers, in particular cationic polymers, such as polyethylenimine (PEI), poly-L-Lysin (PEL), polyvinylamine (PVA) or polyallylamine (PAA), where individual RNA molecules are present in solution. In the formulations, the RNA is preferentially present in the form of monomers, dimers, timers or oligomers, but not as aggregates comprising a large number of RNA molecules per aggregate, in particular large polyplex nanoparticles. The formulations display improved transfection efficacy and they can be used for delivery of RNA to a subject, where they have an improved dose response relationship in comparison to formulations where large aggregates in the form of polyplex nanoparticles are present.

The invention provides saCNTF-DSF-LNP composite nanoparticles as well as a preparation method and application of the saCNTF-DSF-LNP composite nanoparticles, and particularly relates to application of the saCNTF-DSF-LNP to preparation of medicines for treating diseases related to optic nerve injury. The method comprises the following steps: S1, preparing self-amplification CNTF RNA, and naming the self-amplification CNTF RNA as saCNTF; s2, preparing lipid nano particles entrapped with the disulfiram DSF; s3, quickly adding the ethanol phase containing the disulfiram DSF-lipid mixture into the aqueous phase containing the saCNTF, and mixing, so that the nanoparticles are formed by self-assembly; s4, purifying the nano particles by using an ultrafiltration centrifugal device to obtain saCNTF-DSF-LNP; the invention provides a preparation method and application of saCNTF-DSF-LNP, the saCNTF-DSF-LNP can inhibit neuroinflammation driven by acute pyroptosis, and continuous neurotrophic support is provided for RGC survival and long-term axon regeneration; according to saCNTF-DSF-LNP, continuous expression of CNTF is achieved through saRNA, efficient targeted delivery is achieved by means of LNP so as to reduce off-target toxicity of DSF, and a new treatment strategy is provided for neuritis and degenerative diseases.

Disclosed is a compound having a structure of Formula 1. The compound can be used for synthesizing a modified small activating RNA (saRNA) to enhance gene expression modulation activities of saRNA both in vitro and in vivo. A pharmaceutical composition comprising the compound and a method of modulating the expression of a target gene by using the same are also disclosed.

This invention relates to RNA constructs encoding (i) at least one therapeutic biomolecule; and (ii) at least one innate inhibitor protein (IIP). The constructs are RNA replicons and saRNA molecules, and the invention comprises genetic constructs or vectors encoding such RNA replicons. The invention extends to the use of such RNA constructs and replicons in therapy, such as in treating diseases and / or in vaccine delivery. The invention extends to pharmaceutical compositions comprising such RNA constructs, as well as methods and uses thereof.

The present disclosure provides novel self-amplifying RNA (saRNA) constructs that demonstrate enhanced protein expression, prolonged durability, reduced immunogenicity, and the ability to express multiple therapeutic proteins homogeneously. The saRNA constructs comprise a 5' untranslated region (5'UTR), non-structural protein genes derived from alphaviruses, at least one gene of interest encoding a therapeutic protein, a 3' untranslated region (3'UTR), and one or more modified nucleosides. Also disclosed are dual construct systems comprising a first construct encoding non-structural proteins and a second construct encoding one or more genes of interest. Methods of producing and using the saRNA constructs for engineering cells, particularly immune cells, for treatment of various conditions including cancer, inflammatory conditions, and infectious diseases are provided. The saRNA constructs enable the generation of "armored" immune cells expressing multiple therapeutic proteins, thereby providing a multi-pronged approach to complex diseases.

The present application relates to the technical field of biotechnology, in particular to a pseudorabies immunogen composition and application thereof. The present application provides a pseudorabies immunogen composition, which comprises at least one antigen saRNA (self-replicating RNA) molecule; the antigen saRNA molecule comprises at least one open reading frame (ORF), and the open reading frame ORF encodes a polypeptide comprising a gD antigen from a pseudorabies virus. The present application uses the self-replicating RNA technology to prepare a pseudorabies vaccine, and can protect the infection of the pseudorabies virus, which effectively solves the shortcoming of low protection efficiency of the previous pseudorabies vaccine on the pseudorabies virus.

The invention belongs to the technical field of mRNA vaccines, and particularly relates to a self-replicating element, a self-replicating RNA molecule and application thereof. Aiming at the problems that the existing saRNA self-replicating element has cytotoxicity and can induce innate immune response in vivo, so that expression of target gene mRNA (messenger Ribonucleic Acid) is reduced, and further the treatment effect of vaccines and drugs is influenced, the invention provides the self-replicating element which is taken from a VEEVTC83 strain and has the advantages that the self-replicating element can be used for preparing the saRNA self-replicating element; the sequence of the non-structural protein nsP2 of the strain is directionally mutated to be as shown in SEQ ID NO. 1. Meanwhile, the invention also provides a self-replicating RNA molecule, a related vector and a cell. The saRNA vector disclosed by the invention has higher expression quantity and longer expression time on a target gene. The saRNA is used for expressing a tumor vaccine surrounding MUC16, and a vaccine with good prevention and treatment effects on MUC16 positive tumors can be prepared. Therefore, the invention has a good application prospect in the development of medicines and vaccines.

A small activating RNA (saRNA) capable of activating a CCAAT enhancer binding protein alpha (CEBPA) gene, and a delivery system and use thereof are provided, where sense and antisense strands of the saRNA have nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 2, respectively. A biomimetic nano-delivery system for targeted delivery of the saRNA is a biomimetic nanoparticle formed by composite nanoparticle coated by a biomembrane of an inflammatory effector cell, where the composite nanoparticle is formed by histone loading with the saRNA capable of activating the CEBPA gene. The biomimetic nanoparticle can inherit antigens and related membrane functions of the inflammatory effector cell and specifically accumulate at the sites of inflammatory lesions, which enables the biomimetic nanoparticle to not only have a longer circulation time in vivo, but also show inflammatory tropism.

The invention provides a method for effectively concentrating mRNA, mRNA-LNP and saRNA and maintaining the integrity of RNA, the advantages of freeze-drying concentration are adopted, and conditions such as freeze-drying time, freeze-drying initial temperature and freeze-drying temperature are optimized, so that the mRNA-LNP is effectively concentrated, the mRNA concentration is improved, the influence of concentration operation on the integrity of mRNA is reduced, and the method is suitable for large-scale industrial production. The problem that in the preparation and analysis process of an mRNA-LNP preparation, an effective technology is needed for improving the mRNA-LNP concentration is solved. The method is simple and rapid to operate, and has small influence on RNA integrity. By providing a universal and reliable RNA concentration means, the invention aims to overcome the key links of the RNA-LNP preparation from research and development to industrial production and quality detection, and provides key support for the progress of the whole biotechnology industry chain.

The present invention relates to self-amplifying RNA (saRNA) vaccines against infectious laryngotracheitis (ILT) that provide safe and effective prophylactic and therapeutic immune responses and improve the symptoms of the disease.In particular, the invention relates to sRNAs encoding at least one laryngotracheitis virus polypeptide, an ILTV polypeptide variant, or an immunogenic fragment or epitope thereof, said sRNA comprising a first nucleic acid sequence including nsP1, nsP2, nsP3, and nsP4 encoding non-structural alphavirus proteins, a second nucleic acid sequence encoding at least one ILTV polypeptide including gB or gBdel, linked to gD by a nucleic acid sequence encoding at least one protein-binding site or at least one cleavage site for an endogenous or exogenous protease such as a 6k sequence, and also comprising a 5' cap, a 5' UTR upstream of the first nucleic acid sequence, a 26S promoter upstream of the second nucleic acid sequence, a 3' UTR, and a poly tail HAS.The sRNA according to the present invention allows, when incorporated into a cell, the expression of at least one ILTV polypeptide, an ILTV polypeptide variant, or an immunogenic fragment or an epitope of one of them.

The present invention discloses a method for preparing a tumor microenvironment responsive cascade-targeted small activated nucleic acid nanomedicine and its application, and belongs to the field of biomedicine technology. The present invention utilizes the dendritic macromolecule PAMAM modified with M2 type TAM targeting peptide CRVLRSGSC to jointly load the small activated RNA (sa-p38, sa-TFEB) of p38 and TFEB, and uses acid-sensitive liposomes to encapsulate and protect the above-mentioned complex (CRV-PAMAM / saRNA) to prepare tumor microenvironment responsive cascade-targeted small activated nucleic acid nanomedicine, which can reprogram the phenotype and metabolic mode of M2 type TAM to M1 type TAM, thereby killing tumor cells, and remodeling the tumor immune microenvironment, ultimately inhibiting tumor growth and even ablating tumors. The present invention has a very broad application prospect in gene therapy and immunotherapy for cancer, hereditary diseases, infectious diseases, etc.

Self-amplifying RNA (saRNA) vectors are disclosed. The described saRNA vectors are adapted from genotypically diverse alphaviruses. The described saRNA vectors are used in the expression of heterologous genes. Efficacious saRNA vaccines and therapies may be produced from such saRNA vectors.

The present disclosure provides novel self-amplifying RNA (saRNA) constructs that demonstrate enhanced protein expression, prolonged durability, reduced immunogenicity, and the ability to express multiple therapeutic proteins homogeneously. The saRNA constructs comprise a 5' untranslated region (5'UTR), non-structural protein genes derived from alphaviruses, at least one gene of interest encoding a therapeutic protein, a 3' untranslated region (3'UTR), and one or more modified nucleosides. Also disclosed are dual construct systems comprising a first construct encoding non-structural proteins and a second construct encoding one or more genes of interest. Methods of producing and using the saRNA constructs for engineering cells, particularly immune cells, for treatment of various conditions including cancer, inflammatory conditions, and infectious diseases are provided. The saRNA constructs enable the generation of "armored" immune cells expressing multiple therapeutic proteins, thereby providing a multi-pronged approach to complex diseases.

This disclosure provides methods of measuring trans-amplifying RNA and self- amplifying RNA amplification in vivo. The taRNA and the saRNA are barcoded, administered to a subject, and barcode amplification in a specific cell type is measured.

The present invention provides compositions, vaccines, and methods for inducing protective immunity to immunogens in humans. Protective immune responses are obtained by using saRNA, particularly VRP vectors as a prime and MVA vectors for boosting. Specifically, the present invention relates to genetically engineered (recombinant) VRP and MVA vectors that contain at least one heterologous nucleotide sequence encoding an antigenic determinant of an infectious virus (e.g., EBV).

The invention discloses saRNA for activating AMBRA1 transcription, modified saRNA and application of the saRNA and the modified saRNA. AMBRA1 is used as a target spot, saRNA sequences targeting an AMBRA1 promoter region are designed, 31 saRNA sequences and saRNA modified by the saRNA sequences are obtained through screening, and the saRNA sequences and the saRNA modified by the saRNA sequences can up-regulate the expression level of AMBRA1 in tumor cells so as to regulate the cell cycle and inhibit the proliferation, migration and invasion ability of the tumor cells. The invention further studies the feasibility of the saRNA combined chemotherapeutic drug as a tumor combined treatment scheme, and the combined administration of the saRNA and the chemotherapeutic drug further significantly inhibits the proliferation ability of tumor cells, increases apoptosis, and enhances the sensitivity of the tumor cells to the chemotherapeutic drug, so that the prognosis of tumor patients is expected to be improved; the saRNA or the modified saRNA provided by the invention has an application prospect in the aspects of preparation of anti-tumor drugs and preparation of anti-tumor drug combined therapy.

The invention provides a small-amount preparation method of self-replicating RNA (Ribonucleic Acid), which comprises the following steps: (1) preparing an IVT reaction template, (2) carrying out IVT reaction, (3) digesting a DNA (Deoxyribonucleic Acid) template, (4) purifying an IVT sample and (5) carrying out capping reaction: adding a capping buffer solution, GTP (Glutathione Triphosphate), SAM (Soluble Amplified Matrix), an RNA enzyme inhibitor, capping enzyme, 2-O-methoxytransferase and denatured RNA into a reaction container, uniformly mixing, and then incubating; and (6) purification: purifying the sample after the capping reaction. According to the method, the in-vitro transcription and capping process of saRNA is optimized, the method is suitable for small-amount preparation of self-replicating RNA, the operation is simple, the self-replicating RNA with high integrity and normal expression can be obtained, and the system can be amplified.

The invention discloses a GPC3 derived antigen peptide vaccine, an mRNA vaccine, a saRNA vaccine and a preparation method and application thereof. The amino acid sequence of the antigen peptide vaccine is one or any combination of more of amino acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6. According to the invention, an efficient pGPC3 model antigen peptide is screened based on GPC protein, and pGPC3 mRNA and pGPC3 saRNA are designed according to a pGPC3 sequence example. The pGPC3 saRNA (at) Lipi vaccine is prepared through a liposome technology, and due to the fact that limiting conditions such as nonapeptide and high-affinity binding with H-2 I type molecules (H-2Kb) are adopted during pGPC3 polypeptide antigen screening, APC cells are facilitated to present antigens through an MHCI restrictive pathway and activate CD8 + T cells, and GPC3 specific CTL is triggered to efficiently kill liver cancer cells. Finally, the pGPC3 saRNA (at) Lipi vaccine can completely inhibit the growth of the liver cancer, the inhibition rate reaches 99.8%, and the liver cancer of 4 / 8 mice completely fades down. In addition, the method can be used for vaccine adjuvant and carrier research on a liver cancer subcutaneous transplantation tumor model.

The invention discloses application of an EFHD2 gene in preparation of a medicine for treating or preventing myocardial ischemia-reperfusion injury, and relates to the technical field of biological medicines. The applicant obtains expression data of I / R injury related myocardial tissues and normal myocardium from databases such as GEO, and through standardization processing, quality control and difference analysis, and in combination with bioinformatics analysis and experimental verification, the expression of EFHD2 in I / R myocardial cells is obviously reduced. Animal experiments are carried out by constructing a mouse model, specific overexpression of EFHD2 in a mouse heart is carried out by using a saRNA vector, and it is found that the saRNA vector can significantly improve the cardiac function after myocardial ischemia-reperfusion, reduce the cardiac infarction area after myocardial ischemia-reperfusion and improve myocardial cell apoptosis after myocardial ischemia-reperfusion; the invention reveals that EFHD2 can improve myocardial ischemia reperfusion injury for the first time, provides a new drug action target for prevention and treatment of myocardial ischemia reperfusion injury, and has very important clinical transformation value.