Systems, methods, and compositions for site-specific genetic engineering using programmable addition via site-specific targeting elements (paste)

12195,733

18/487610

October 16, 2023

This disclosure provides systems, methods, and compositions for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE). PASTE comprises the addition of an integration site into a target genome followed by the insertion of one or more genes of interest or one or more nucleic acid sequences of interest at the site. PASTE combines gene editing technologies and integrase technologies to achieve unidirectional incorporation of genes in a genome for the treatment of diseases and diagnosis of disease.

Massachusetts Institute of Technology (Cambridge, MA, US)

Massachusetts Institute of Technology (Cambridge, MA, US)

1. A system capable of site-specifically integrating an exogenous nucleic acid into a mammalian cell genome at a desired target site, wherein the system comprises, concurrently within the mammalian cell: (a) a nucleic acid encoding a DNA binding nickase domain linked to a reverse transcriptase domain, wherein the linked nickase-RT domains are further linked to a serine integration enzyme via a linker selected from P2A, (GGGS) 3 , GGGGS, PAPAP, (EAAAK) 3 , XTEN, (GGS) 6 , and EAAAK; (b) a guide RNA or a nucleic acid encoding a guide RNA (gRNA), the guide RNA comprising, from 3′ to 5′, i. a primer binding sequence, ii. a sequence complementary to one strand of an AttB or AttP integration recognition sequence, and iii. a target binding sequence, wherein the gRNA is capable of guiding the linked nickase-reverse transcriptase domains to the genomic target site; (c) an exogenous nucleic acid linked to a sequence that is an integration cognate of the integration recognition sequence, whereby the system site specifically integrates the exogenous nucleic acid into the mammalian cell genome at the desired target site.

2. The system of claim 1 , wherein the DNA binding nickase domain is linked to the reverse transcriptase domain by in-frame fusion.

3. The system of claim 1 , wherein the DNA binding nickase domain is linked to the reverse transcriptase domain by a linker.

4. The system of claim 3 , wherein the linker is a peptide fused in-frame between the nickase and reverse transcriptase domains.

5. The system of claim 1 , wherein the DNA binding nickase domain is selected from Cas9-D10A, Cas9-H840A, and Cas12a/b nickase.

6. The system of claim 1 , wherein the reverse transcriptase domain is selected from the group consisting of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV-RT), and Eubacterium rectale maturase RT.

7. The system of claim 6 , wherein the reverse transcriptase domain is a M-MLV reverse transcriptase domain.

8. The system of claim 6 , wherein the M-MLV reverse transcriptase domain comprises one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W.

9. The system of claim 1 , wherein the exogenous nucleic acid is a minicircle, a plasmid, a mRNA, or a linear DNA.

10. The system of claim 8 , wherein exogenous nucleic acid is a minicircle.

11. The system of claim 10 , wherein the minicircle does not comprise a sequence of a bacterial origin.

12. The system of claim 1 , wherein the serine integration enzyme is selected from the group consisting of Bxb1, φC31, RDF, φBT1, R1, R2, R3, R4, RS, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, and φRV.

13. The system of claim 12 , wherein the serine integration enzyme is Bxb1.

14. The system of claim 1 , wherein the exogenous nucleic acid encodes: a reporter gene; a degradation tag for programmable knockdown of proteins in the presence of small molecules; a T-cell receptor (TCR), a chimeric antigen receptor (CAR), an interleukin, a cytokine, or an immune checkpoint gene and the mammalian cell is a T-cell or natural killer (NK) cell; a beta hemoglobin (HBB) gene and the mammalian cell is a hematopoietic stem cell (HSC); a metabolic gene; or a gene involved in an inherited disease or syndrome.

15. The system of claim 1 , wherein the exogenous nucleic acid is between 1000 bp and 36,000 bp in length.

16. The system of claim 1 , wherein the exogenous nucleic acid is more than 36,000 bp in length.

17. The system of claim 1 , wherein the exogenous nucleic acid is less than 1000 bp in length.

18. The system of claim 14 , wherein the inherited disease is cystic fibrosis, familial hypercholesterolemia, adenosine deaminase (ADA) deficiency, X-linked SCID (X-SCID), Wiskott-Aldrich syndrome (WAS), hemochromatosis, Tay-Sachs, fragile X syndrome, Huntington's disease, Marfan syndrome, phenylketonuria, or muscular dystrophy.

19. The system of claim 1 , further comprising a nicking gRNA.

20. The system of claim 1 , wherein (a)-(c) are introduced into the mammalian cell as an adeno-associated virus (AAV) or an adenovirus (AdV).

21. The system of claim 1 , wherein (a)-(c) are delivered in a single transfection.

22. The system of claim 1 , wherein (a)-(c) are co-expressed.

23. The system of claim 1 , wherein the linker comprises P2A.

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