# US 12,006,525 B2 · Production of RNA by yeasts with recombinant pseudo-viral particles

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- Number: US 12,006,525 B2
- Document ID: US12006525B2
- Status: Granted
- Kind: B2
- Application number: 16/495,874
- Filed: 2018-03-21
- Published: 2020-01-09
- Granted: 2024-06-11
- Earliest claimed: 2017-03-21
- Assignees: Centre National De La Recherche Scientifique; Universite D'Orleans
- Applicants: Centre National De La Recherche Scientifique; Universite D'Orleans
- Inventors: Lucie Pigeon; Rachid A. Rahmouni; Harivony Chantal Pichon-Rabenandrasana; Patrick Midoux
- Web page: https://patentgrep.com/patent/US12006525B2

## CPC

- C07K 14/39: from yeasts
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- C07K 14/395: from Saccharomyces
- C12N 9/1247: {DNA-directed RNA polymerase (2.7.7.6)}
- C12N 15/81: for yeasts
- C12Y 207/07006: DNA-directed RNA polymerase (2.7.7.6)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The application relates to the production of RNA of interest, more specifically of messenger RNA of interest or of long non-coding RNA of interest, by yeasts with recombinant pseudo-viral particles. The recombinant yeasts have been genetically modified in order to produce the RNA of interest in the form of a complex, particularly in the form of recombinant pseudo-viral particles. These recombinant pseudo-viral particles are produced from certain elements of yeast Ty retrotransposon, but do not retrotranscribe the RNA that they contain. Thus, the application relates to the components that are thus capable of being implemented or produced, and particularly to the nucleic acid constructs, kits, bacteria cells, yeast cells, culture or transfection media containing them, as well as to a method for producing a pharmaceutical composition, particularly for medical applications, more specifically for vaccines, anti-tumour and pro-regenerative applications.

## Claims

### Claim 1

A method for the recombinant production of messenger RNA (mRNA) or non-coding long-chain RNA (lncRNA), which comprises:
culturing yeast cells, which have been genetically modified to produce recombinant pseudo-viral particles which comprise said mRNA or lncRNA, and
collecting the mRNA or lncRNA which is contained in the virus-like particles thus formed,
wherein the yeast cells are cells of a yeast which is free of Ty1 retrotransposon or in which any Ty1 retrotransposon has been genetically modified to not produce a Ty1 retrosome,
wherein the genetic modifications made to these yeast cells to produce said virus-like particles comprise transfection of these yeast cells by
i. a nucleic acid comprising a first nucleotide sequence, and
ii. a nucleic acid comprising a second nucleotide sequence,
wherein:
the nucleic acid of i. and the nucleic acid of ii. are carried by the same molecule, or by two distinct or separate molecules,
the first nucleotide sequence comprises a DNA sequence which encodes the expression of the Gag protein of a yeast Ty1 retrotransposon,
the second nucleotide sequence comprises a DNA sequence having no reading frame, the first codon of which would be the ATG codon, whose RNA transcript contains two parts:
a. the sequence of said mRNA or lncRNA linked to
b. the gag addressing sequence comprising the sequence of SEQ ID NO: 16,
the nucleic acid of ii. does not comprise a DNA sequence whose RNA transcript would be the complete RNA sequence of said Gag protein of a yeast Ty1 retrotransposon,
the Gag protein of a yeast Ty retrotransposon in both nucleic acids is the same Gag protein of a yeast Ty1 retrotransposon, and
the nucleic acid of i. and the nucleic acid of ii. do not comprise a sequence encoding the reverse transcriptase of a yeast Ty1 retrotransposon, the nucleic acid of i. and the nucleic acid of ii. thereby encoding the formation, in said yeast cells, of virus-like particles which are formed by the Gag protein translated from the first nucleotide sequence, and which encapsulate said mRNA or lncRNA transcribed from the second nucleotide sequence.

### Claim 2 (depends on 1)

The method of claim 1, wherein the yeast is a yeast of the genus Saccharomyces or Pichia.

### Claim 3 (depends on 1)

The method of claim 1, wherein the yeast is a yeast of which one or more of the Rpb1, Spt21, Srb2 and Srb5 genes have been mutated by one or more mutations that comprise one or more mutations selected from:
replacing one or more codons each with another codon or with another nucleotide triplet, and
deleting one or more nucleotides,
and which stimulate or increase the production of pseudo-viral particles.

### Claim 4 (depends on 1)

The method of claim 1, wherein the mRNA or lncRNA comprises at least 150 nucleotides and is heterologous to said yeast, and wherein the mRNA or lncRNA optionally encodes:
a bacteria protein or a polypeptide,
a genomic editing enzyme,
a virus protein or polypeptide,
a human transcription factor,
a human growth factor,
a human CFTR protein,
an antibody or antibody fragment, or
a polypeptide comprising at least one epitope of antigens.

### Claim 5 (depends on 1)

The method of claim 1, performed In vitro using a kit comprising at least one nucleic acid comprising a first nucleotide sequence and at least one nucleic acid comprising a second nucleotide sequence, wherein:
the first nucleotide sequence comprises a DNA sequence which encodes the expression of the Gag protein of a yeast Ty1 retrotransposon,
the second nucleotide sequence comprises a DNA sequence which comprises the gag addressing sequence comprising the sequence of SEQ ID NO: 16, the DNA sequence of this second nucleotide sequence that does not comprise a reading frame whose first codon would be the ATG codon,
the nucleic acid that comprises the second nucleotide sequence does not comprise a DNA sequence whose RNA transcript would be the complete RNA sequence of said retrotransposon Gag protein Ty1 of yeast, and
the nucleic acid which comprises the first nucleotide sequence and the nucleic acid which comprises the second nucleotide sequence are carried by the same molecule, or are carried by two distinct or separate molecules,
said kit comprising no reverse transcriptase of yeast Ty1 retrotransposon, nor nucleic acid encoding such a reverse transcriptase, nor nucleic acid comprising a sequence encoding such a reverse transcriptase, and
said kit may further optionally comprise cells of a yeast which is devoid of Ty1 retrotransposon, or that has been genetically modified to not produce a Ty1 retrosome,
wherein the method is performed for the production of mRNA or lncRNA with a polyA tail at the 3′ end and/or a 7-methylguanosine cap at the 5′ end.

### Claim 6 (depends on 1)

A method for producing a pharmaceutical composition which comprises at least one mRNA or lncRNA, said method comprising producing mRNA or lncRNA by the method of claim 1, and placing the mRNA or lncRNA in a pharmaceutically acceptable vehicle for producing a pharmaceutical composition, this pharmaceutical composition being optionally intended for:
the prevention or the treatment of a microbiological infection,
the prevention or the treatment of a tumor proliferation,
the prevention or the treatment of a chronic disease,
a regenerative tissue or cell therapy, or
a gene therapy.

### Claim 7 (depends on 2)

The method of claim 2, wherein the yeast is a yeast of which one or more of the Rpb1, Spt21, Srb2 and Srb5 genes have been mutated by one or more mutations that comprise one or more mutations selected from:
replacing one or more codons each with another codon or with another nucleotide triplet, and
deleting one or more nucleotides,
and which stimulate or increase the production of pseudo-viral particles.

### Claim 8 (depends on 2)

The method of claim 2, wherein the mRNA or lncRNA comprises at least 150 nucleotides and is heterologous to said yeast, and wherein the mRNA or lncRNA optionally encodes:
a bacteria protein or a polypeptide,
a genomic editing enzyme,
a virus protein or polypeptide,
a human transcription factor,
a human growth factor,
a human CFTR protein,
an antibody or antibody fragment, or
a polypeptide comprising at least one epitope of antigens.

### Claim 9 (depends on 3)

The method of claim 3, wherein the mRNA or lncRNA comprises at least 150 nucleotides and is heterologous to said yeast, and wherein the mRNA or lncRNA optionally encodes:
a bacteria protein or a polypeptide,
a genomic editing enzyme,
a virus protein or polypeptide,
a human transcription factor,
a human growth factor,
a human CFTR protein,
an antibody or antibody fragment, or
a polypeptide comprising at least one epitope of antigens.