# US 12,180,473 B2 · Chimeric RNA-driven genomic rearrangement in mammalian cells

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- Number: US 12,180,473 B2
- Document ID: US12180473B2
- Status: Granted
- Kind: B2
- Application number: 17/047,924
- Filed: 2019-04-16
- Published: 2021-08-05
- Granted: 2024-12-31
- Earliest claimed: 2018-04-16
- Assignees: Baylor College of Medicine
- Applicants: Baylor College of Medicine
- Inventors: Laising Yen; Sachin Kumar Gupta; Jocelyn Duen-Ya Jea
- Web page: https://patentgrep.com/patent/US12180473B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 2310/11: Antisense
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Embodiments of the disclosure include methods and compositions in which two inter-chromosomal or intra-chromosomal genomic DNA regions are rearranged upon exposure of the genomic DNA to a chimeric RNA that produces fusion of the two regions. The chimeric RNA hybridizes to the two different parts of the genomic DNA and forms a DNA/RNA hybrid in a sequence-specific manner, thereby facilitating genomic rearrangement. In particular embodiments this mechanism is utilized to produce directed gene fusion in targeted genomic DNA regions.

## Claims

### Claim 1

A method of producing a genomic fusion product through endogenous genomic DNA rearrangements between a first and a second region of genomic DNA in a cell, in vitro, comprising the step of: exposing to the genomic DNA an effective amount of an exogenously provided synthetic chimeric RNA, said chimeric RNA comprising (1) a first RNA sequence that is reverse complement with respect to a portion of the first region of genomic DNA and (2) a second RNA sequence that is reverse complement with respect to a portion of the second region of genomic DNA, wherein the first RNA sequence is between 30-150 nucleotides long and the second RNA sequence is between 30-150 nucleotides long, wherein a portion of the first genomic region that is not reverse complement to the first RNA sequence is reverse complementary to a portion of the second genomic region that is not reverse complement to the second RNA sequence, and wherein upon exposing of the chimeric RNA to the genomic DNA, the first and second genomic regions become fused to each other thereby producing the genomic fusion product through endogenous DNA recombination.

### Claim 2 (depends on 1)

The method of claim 1, wherein upon exposing the chimeric RNA to the genomic DNA, the chimeric RNA hybridizes with the complementary portions of the first and second regions of genomic DNA through the first and second RNA sequences of the chimeric RNA, thereby forming a DNA/RNA hybrid in a sequence-specific manner.

### Claim 3 (depends on 2)

The method of claim 2, wherein upon hybridization of the chimeric RNA with the respective complementary sequences of the first and second genomic regions of the genomic DNA, a double stranded DNA/DNA stem structure is produced between the portions of genomic DNA that are complementary to each other.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first and second regions of genomic DNA are on the same chromosome.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first and second regions of genomic DNA are on different chromosomes.

### Claim 6 (depends on 1)

The method of claim 1, wherein at least one of the first and second regions of genomic DNA has active transcription.

### Claim 7 (depends on 6)

The method of claim 6, wherein the chimeric RNA is antisense with respect to the at least one region having active transcription.

### Claim 8 (depends on 1)

The method of claim 1, wherein at least one of the first and second regions of genomic DNA lacks transcriptional activity.

### Claim 9 (depends on 8)

The method of claim 8, wherein one or both of the first RNA sequence and second RNA sequence of the chimeric RNA is either antisense or sense with respect to the at least one of the two different regions lacking active transcription.

### Claim 10 (depends on 1)

The method of claim 1, wherein the chimeric RNA has or is designed to have between 27% and 65% GC content.

### Claim 11 (depends on 1)

The method of claim 1, wherein the cell is a diseased cell.

### Claim 12 (depends on 1)

The method of claim 1, wherein the method occurs at physiological hormone levels.

### Claim 13 (depends on 1)

The method of claim 1, wherein the synthetic chimeric RNA is produced in a cell.

### Claim 14 (depends on 3)

The method of claim 3, wherein the DNA region between the DNA stem structure and the DNA/RNA hybrid in the first genomic region is between 15-50 nucleotides long and wherein the DNA region between the DNA stem structure and the DNA/RNA hybrid in the second genomic region is between 15-50 nucleotides long.

### Claim 15 (depends on 14)

The method of claim 14, wherein the DNA region between the DNA stem structure and the DNA/RNA hybrid in the first genomic region is single stranded.

### Claim 16 (depends on 14)

The method of claim 14, wherein the DNA region between the DNA stem structure and the DNA/RNA hybrid in the second genomic region is single stranded.