Lentiviral vector and method for delivering exogenous RNA by the lentiviral vector

Abstract

A lentiviral vector and a method for delivering an exogenous RNA by the lentiviral vector are provided. The lentiviral vector is prepared by transfecting plasmids containing a genome sequence of the lentiviral vector into a virus-producing cell, collecting a supernatant and concentration. Specifically, according to the principle of combining an RNA-binding protein with an RNA sequence identified by the RNA-binding protein, the RNA-binding protein is integrated into a skeleton of a lentivirus GagPol long-chain protein, and the RNA sequence identified by the RNA-binding protein is connected to the exogenous target RNA, so that the exogenous target RNA is packaged into lentiviral particles during the assembly of the lentiviral particles. The exogenous target RNA can be mRNA, gRNA or RNA with other functions. The present invention can be used in the fields of gene editing, gene therapy, cell therapy, immunotherapy, regenerative medicine and basic biology.

Claims

1. A lentiviral vector, wherein the lentiviral vector is prepared by transfecting plasmids containing a genome sequence of the lentiviral vector into a virus-producing cell, collecting a first supernatant and concentrating the first supernatant; the plasmids comprise a plasmid expressing an envelope protein, a plasmid expressing a lentivirus GagPol long-chain protein containing an RNA-binding protein and a plasmid containing exogenous target RNA with an RNA stem-loop structure identified by the RNA-binding protein; wherein the plasmid expressing the lentivirus GagPol long-chain protein containing the RNA-binding protein integrates the RNA-binding protein into an N-terminal of a third-generation lentivirus GagPol long-chain protein, and the plasmid expressing the envelope protein is VSV-G; wherein a codon sequence of the lentivirus GagPol long-chain protein is SEQ ID NO: 1; and wherein the RNA-binding protein is MS2 coat protein, and a codon sequence of the MS2 coat protein is SEQ ID NO: 2.

2. The lentiviral vector according to claim 1, wherein in the plasmid containing the RNA stem-loop structure, the RNA stem-loop structure is an RNA sequence identified and bound by the RNA-binding protein, and wherein the RNA sequence is SEQ ID NO: 3.

3. The method according to claim 1, wherein the virus-producing cell is one selected from the group consisting of 293T, 293FT and HEK293.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Other features, objectives and advantages of the present invention will become more apparent upon reading the detailed description of non-restrictive embodiments with reference to the following drawings:

(2) FIG. 1 is a diagram showing an experiment of a lentiviral vector delivering GFP mRNA; and

(3) FIG. 2 is a diagram showing an experiment of a lentiviral vector delivering Cas9 mRNA for gene editing.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(4) The present invention is described in detail below in combination with the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but will not limit the present invention in any form. It should be noted that numerous modifications and improvements may be made by those skilled in the art without departing from the spirit of the present invention. These modifications and improvements are within the protection scope of the present invention.

Embodiment 1: Preparation of Lentiviral Particles

(5) Step 1: a plasmid expressing a envelope protein, a plasmid expressing a lentivirus GagPol long-chain protein containing a RNA-binding protein and a plasmid expressing an exogenous target RNA containing an RNA stem-loop structure identified by the RNA-binding protein are co-transfected into virus-producing cells (293T). 1) The plasmid expressing the envelope protein includes VSV-G, CD4 recognition protein, CD8 recognition protein, RD114 and baboon endogenous retrovirus envelope protein-modified envelope protein. VSV-G is selected in the present embodiment. 2) The virus-producing cells include 293T, 293FT, HEK293, etc. 293T is selected in the present embodiment. 3) The plasmid expressing the lentivirus GagPol long-chain protein containing the RNA-binding protein is obtained by fusing an MS2 coat protein with the lentivirus GagPol long-chain protein in the present embodiment.

(6) A codon sequence of the GagPol long-chain protein is as follows (SEQ ID NO: 1):

(7) TABLE-US-00001 SEQIDNO.1 gccagggccagcgtgctgagcggcggcgagctggacaggtgggagaagat caggctgaggcccggcggcaagaagaagtataagctgaagcacatcgtgt gggccagcagggagctggagaggttcgccgtgaaccccggcctgctggag accagcgagggctgcaggcagatcctgggccagctgcagcccagcctgca gaccggcagcgaggagctgaggagcctgtacaacaccgtggccaccctgt actgcgtgcaccagaggatcgagatcaaggacaccaaggaggccctggac aagatcgaggaggagcagaacaagtccaagaagaaggcccagcaggccgc cgccgacaccggccacagcagccaggtgagccagaactaccccatcgtgc agaacatccagggccagatggtgcaccaggccatcagccccaggaccctg aacgcctgggtgaaggtggtggaggagaaggccttcagccccgaggtgat ccccatgttcagcgccctgagcgagggagccaccccccaggacctgaaca ccatgctgaacaccgtgggcggccaccaggccgccatgcagatgctgaag gagaccatcaacgaggaggccgccgagtgggacagggtgcaccccgtgca cgccggccccatcgcccccggccagatgagggagccccgcggcagcgaca tcgccggcaccaccagcaccctgcaggagcagatcggctggatgaccaac aacccccccatccccgtgggcgaaatctacaagaggtggatcatcctggg cctgaacaagatcgtgaggatgtacagccccaccagcatcctggatatca ggcagggccccaaagagcccttcagggactacgtggacaggttctacaag accctgcgcgccgagcaggccagccaggaggtgaagaactggatgaccga gaccctgctggtgcagaacgccaaccccgactgcaagaccatcctgaagg ccctgggacccgccgccaccctggaggagatgatgaccgcctgccagggc gtgggcggccccggccacaaggccagggtgctggccgaggccatgagcca ggtgaccaacaccgccaccatcatgatgcagaggggcaacttcaggaacc agaggaagatggtgaagtgcttcaactgcggcaaggagggccacaccgcc aggaactgccgcgcccccaggaagaagggctgctggaagtgcggcaagga gggccaccagatgaaggactgcaccgagaggcaggctaattttttaggga agatctggccttcctacaagggaaggccagggaattttcttcagagcaga ccagagccaacagccccaccatttcttcagagcagaccagagccaacagc cccaccagaagagagcttcaggtctggggtagagacaacaactccccctc agaagcaggagccgatagacaaggaactgtatcctttaacttccctcaga tcactctttggcaacgacccctcgtcacaataaagatcggtggccagctg aaggaggccctgctggacaccggcgccgacgacaccgtgctggaggagat gagcctgcccggcaggtggaagcccaagatgatcggcggcatcggcggct tcatcaaggtgaggcagtacgaccagatcctgatcgagatctgcggccac aaggccatcggcaccgtgctggtgggacctacacctgtgaacatcatcgg caggaacctgctgacccagatcggctgcaccctgaacttccccatcagcc ccatcgagaccgtgcccgtgaagctgaagcccggcatggacggccctaag gtgaagcagtggcccctgaccgaggagaagatcaaggccctggtggagat ctgcaccgagatggagaaggagggcaagatcagcaagatcggccccgaga acccctacaacacccccgtgttcgccatcaagaagaaggacagcaccaag tggaggaagctggtggacttcagggagctgaacaagaggacccaggactt ctgggaggtgcagctgggcatcccccaccccgccggcctgaagaagaaga agagcgtgaccgtgctggacgtgggcgacgcctacttcagcgtgcccctg gacgaggacttcaggaagtataccgccttcaccatccccagcatcaacaa cgagacccccggcatccgctaccagtacaacgtgctgccccagggctgga agggcagccccgccatcttccagagcagcatgacaaagatcctggagccc ttcaagaagcagaaccccgacatcgtgatctatcagtacatggacgacct gtacgtgggcagcgacctggagatcggccagcacaggaccaagatcgagg agctgaggcagcacctgctgaggtggggcctgaccacccccgacaagaag caccagaaggagcccccattcctgtggatgggctacgagctgcaccccga caagtggaccgtgcagcccatcgtgctgcccgagaaggacagctggaccg tgaacgacattcagaagctggtgggcaagctgaactgggccagccagatc taccccggcatcaaggtgaggcagctgtgcaagctgctgaggggcacaaa ggctctgaccgaggtgatccccctgaccgaggaggccgagctggagctgg ccgagaacagggagatcctgaaggagcccgtgcacggcgtgtactacgac cccagcaaggacctgatcgccgagatccagaagcagggccagggccagtg gacctaccagatctaccaggagcccttcaagaacctgaagaccggcaagt acgcccgcatgcgcggcgcccacaccaacgacgtgaagcagctgaccgag gccgtgcagaagatcaccaccgagagcatcgtgatctggggcaagactcc taagttcaagctgcccatccagaaggagacctgggagacctggtggaccg agtactggcaggccacctggattcccgagtgggagttcgtgaacacccct cccctggtgaagctgtggtatcagctggagaaggagcccatcgtgggcgc cgagaccttctacgtggacggcgccgccaacagggagaccaagctgggca aggccggctacgtgaccaacaagggccgccagaaggtggtgcccctgacc aacaccaccaaccagaagaccgagctgcaggctatctacctggccctgca ggactcaggcctggaggtgaacatcgtgaccgacagccagtacgccctgg gcatcatccaggcccagcccgacaagagcgagagcgagctggtgaaccag atcatcgagcagctgatcaagaaggagaaggtgtacctggcctgggtgcc cgcccacaagggcatcggcggcaacgagcaggtggacaagctggtgagcg ccggcatcaggaagatcctgttcctggacggcatcgacaaggcccaggac gagcacgagaagtaccacagcaactggagggctatggctagcgacttcaa cctgcctcccgtggtggctaaggagatcgtggccagctgcgacaagtgcc agctgaagggcgaggccatgcacggccaggtggactgcagccccggcatc tggcagctggtttgcacccacctggagggcaaggtgatcctggtggccgt gcacgtggcctccggctacatcgaggccgaggtgatccccgccgagaccg gccaggagaccgcctacttcctgctgaagctggccggccgctggcccgtg aagaccatccacaccgacaacggcagcaacttcaccagcgccaccgtgaa ggccgcctgctggtgggccggcatcaagcaggagttcggcatcccctaca acccccagtctcagggcgtggtggagagcatgaacaaggagctgaagaag atcatcggccaggtgagggaccaggccgagcacctgaagaccgccgtgca gatggccgtgttcatccacaacttcaagaggaagggcggcatcggcggct acagcgccggcgagaggatcgtggacatcatcgccaccgacatccagacc aaggagctgcagaagcagatcaccaagatccagaacttcagggtgtacta cagggacagcaggaaccctctgtggaagggccccgccaagctgctgtgga agggcgagggcgccgtggtgatccaggacaacagcgacatcaaggtggtg cccaggaggaaggccaagatcatcagggactacggcaagcagatggccgg cgacgactgcgtggcctccaggcaggacgaggactga.
accagagccaacagccccaccatttcttcagagcagaccagagccaacagccccaccagaagagagcttcaggtctggggtagaga caacaactccccctcagaagcaggagccgatagacaaggaactgtatcctttaacttccctcagatcactctttggcaacgacccctcgt cacaataaagatcggtggccagctgaaggaggccctgctggacaccggcgccgacgacaccgtgctggaggagatgagcctgccc ggcaggtggaagcccaagatgatcggcggcatcggcggcttcatcaaggtgaggcagtacgaccagatcctgatcgagatctgcgg ccacaaggccatcggcaccgtgctggtgggacctacacctgtgaacatcatcggcaggaacctgctgacccagatcggctgcaccct gaacttccccatcagccccatcgagaccgtgcccgtgaagctgaagcccggcatggacggccctaaggtgaagcagtggcccctga ccgaggagaagatcaaggccctggtggagatctgcaccgagatggagaaggagggcaagatcagcaagatcggccccgagaacc cctacaacacccccgtgttcgccatcaagaagaaggacagcaccaagtggaggaagctggtggacttcagggagctgaacaagag gacccaggacttctgggaggtgcagctgggcatcccccaccccgccggcctgaagaagaagaagagcgtgaccgtgctggacgtg ggcgacgcctacttcagcgtgcccctggacgaggacttcaggaagtataccgccttcaccatccccagcatcaacaacgagaccccc ggcatccgctaccagtacaacgtgctgccccagggctggaagggcagccccgccatcttccagagcagcatgacaaagatcctgga gcccttcaagaagcagaaccccgacatcgtgatctatcagtacatggacgacctgtacgtgggcagcgacctggagatcggccagca caggaccaagatcgaggagctgaggcagcacctgctgaggtggggcctgaccacccccgacaagaagcaccagaaggagcccc cattcctgtggatgggctacgagctgcaccccgacaagtggaccgtgcagcccatcgtgctgcccgagaaggacagctggaccgtg aacgacattcagaagctggtgggcaagctgaactgggccagccagatctaccccggcatcaaggtgaggcagctgtgcaagctgct gaggggcacaaaggctctgaccgaggtgatccccctgaccgaggaggccgagctggagctggccgagaacagggagatcctgaa ggagcccgtgcacggcgtgtactacgaccccagcaaggacctgatcgccgagatccagaagcagggccagggccagtggaccta ccagatctaccaggagcccttcaagaacctgaagaccggcaagtacgcccgcatgcgcggcgcccacaccaacgacgtgaagcag ctgaccgaggccgtgcagaagatcaccaccgagagcatcgtgatctggggcaagactcctaagttcaagctgcccatccagaagga gacctgggagacctggtggaccgagtactggcaggccacctggattcccgagtgggagttcgtgaacacccctcccctggtgaagct gtggtatcagctggagaaggagcccatcgtgggcgccgagaccttctacgtggacggcgccgccaacagggagaccaagctgggc aaggccggctacgtgaccaacaagggccgccagaaggtggtgcccctgaccaacaccaccaaccagaagaccgagctgcaggct atctacctggccctgcaggactcaggcctggaggtgaacatcgtgaccgacagccagtacgccctgggcatcatccaggcccagcc cgacaagagcgagagcgagctggtgaaccagatcatcgagcagctgatcaagaaggagaaggtgtacctggcctgggtgcccgcc cacaagggcatcggcggcaacgagcaggtggacaagctggtgagcgccggcatcaggaagatcctgttcctggacggcatcgaca aggcccaggacgagcacgagaagtaccacagcaactggagggctatggctagcgacttcaacctgcctcccgtggtggctaaggag atcgtggccagctgcgacaagtgccagctgaagggcgaggccatgcacggccaggtggactgcagccccggcatctggcagctgg tttgcacccacctggagggcaaggtgatcctggtggccgtgcacgtggcctccggctacatcgaggccgaggtgatccccgccgag accggccaggagaccgcctacttcctgctgaagctggccggccgctggcccgtgaagaccatccacaccgacaacggcagcaactt caccagcgccaccgtgaaggccgcctgctggtgggccggcatcaagcaggagttcggcatcccctacaacccccagtctcagggc gtggtggagagcatgaacaaggagctgaagaagatcatcggccaggtgagggaccaggccgagcacctgaagaccgccgtgcag atggccgtgttcatccacaacttcaagaggaagggcggcatcggcggctacagcgccggcgagaggatcgtggacatcatcgccac cgacatccagaccaaggagctgcagaagcagatcaccaagatccagaacttcagggtgtactacagggacagcaggaaccctctgt ggaagggccccgccaagctgctgtggaagggcgagggcgccgtggtgatccaggacaacagcgacatcaaggtggtgcccagga ggaaggccaagatcatcagggactacggcaagcagatggccggcgacgactgcgtggcctccaggcaggacgaggactga SEQ ID NO: 1.

(8) A codon sequence of the MS2 coat protein is as follows (SEQ ID NO: 2), and its encoded protein can be linked to the GagPol long-chain protein encoded by the sequence of SEQ ID NO: 1, for example, the protein encoded by the MS2 coat protein is placed on the N-terminal of the GagPol long-chain protein.

(9) TABLE-US-00002 SEQIDNO.2 atggcctctaattttactcaatttgtgcttgtcgataatggggggacggg agatgtgaccgttgcccctagcaatttcgcaaatggcgttgcagaatgga tctctagcaacagcagaagccaagcgtacaaagtaacgtgttccgttcgc caaagctccgcccaaaaacggaagtatacaataaaggttgaggtgccgaa agtagccactcaaacagttggtggggtagaattgcccgtagcggcatggc ggtcatatctcaatatggaactcactatcccaatcttcgccacgaatagc gattgtgagctgatagttaaggctatgcaaggtcttctcaaagatggaaa ccctattccatctgctatcgccgccaacagcgggatatac. 4) The plasmid expressing the exogenous target RNA containing the RNA stem-loop structure identified by the RNA-binding protein is obtained by fusing a sequence of an RNA identified by the MS2 coat protein with a sequence of the target RNA.

(10) The sequence of the RNA identified by the MS2 coat protein is as follows (SEQ ID NO: 3), which can be linked with the exogenous target RNA by single or multiple repeats.

(11) TABLE-US-00003 SEQIDNo.3 ACAUGAGGAUCACCCAUGU. 5) The plasmids for preparing lentiviral particles carrying the exogenous target RNA mentioned in 3) and 4) above are obtained by a molecular cloning method. Step 2: a supernatant containing viral particles is collected and concentrated by a high-speed centrifugation or a HPLC method to obtain the lentiviral particles containing the exogenous target RNA with a high titer.

Embodiment 2: Delivery of GFP mRNA Using a Lentiviral Vector

(12) The specific steps are the same as those in embodiment 1. GFP mRNA is selected as an exogenous target RNA. Green fluorescent protein (GFP) is a fluorescent protein as a reporter gene. The present embodiment specifically delivers the GFP mRNA into 293T cells by a lentiviral vector.

(13) As shown in FIG. 1, after the 293T cells are infected with lentiviral particles carrying the GFP mRNA for 48 h, GFP positive cells reached 99.8% by flow cytometry.

Embodiment 3: Delivery of Cas9 mRNA and gRNA Targeting AAVS1 Site by a Lentiviral Vector

(14) The specific steps are the same as those in embodiment 1. Cas9 mRNA and gRNA targeting AAVS1 site are selected as exogenous target RNAs. The present embodiment specifically delivers the Cas9 mRNA and the gRNA targeting the AAVS1 site of human