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US-20260125669-A1 - METHODS AND COMPOSITIONS FOR SYNTHETIC EVOLUTION

US20260125669A1US 20260125669 A1US20260125669 A1US 20260125669A1US-20260125669-A1

Abstract

A method of synthetic evolution of a nucleic acid sequence of interest and the mutant nucleic acid sequence of interest obtained therefrom. The method utilizes a virus-assisted evolution platform for hyper-directed evolution of phenotypes in mammalian cells. The virus-assisted evolution platform includes co-transfected mammalian producer cells with at least a first nucleic acid sequence encoding a Retroviridae genome including at least the gag and pol genes and a at least a second nucleic acid encoding a recombinant expression cassette.

Inventors

  • Dimitrije IVANCIC
  • Marc GÜELL CARGOL

Assignees

  • UNIVERSITAT POMPEU FABRA

Dates

Publication Date
20260507
Application Date
20231006
Priority Date
20221007

Claims (20)

  1. 1 - 15 . (canceled)
  2. 16 . A method of synthetic evolution of a nucleic acid sequence of interest (SoI), comprising: (a) co-transfecting mammalian producer cells with (i) a first nucleic acid sequence encoding a Retroviridae genome comprising at least the gag and pol genes, (ii) a second nucleic acid sequence encoding a recombinant expression cassette comprising, from 5′ to 3′: a 5′ long terminal repeat (5′ LTR), a packaging sequence, the SoI to be evolved, a 3′ long terminal repeat (3′ LTR); (b) incubating the mammalian producer cells under conditions allowing for mutation of the SoI and production of Retroviridae vectors; (c) harvesting the population of Retroviridae vectors produced after step (b); (d) infecting mammalian reporter cells with the population of Retroviridae vectors of (c); (e) incubating the mammalian reporter cells under conditions allowing for SoI expression; and (f) selecting one or several mutants of the SoI with a desired biological activity; wherein steps (a)-(f) are reiterated until a mutant of the SoI with the desired biological activity is obtained.
  3. 17 . The method according to claim 16 , wherein at step (a) said mammalian producer cells are further co-transfected with (iii) a third nucleic acid sequence encoding a viral envelope glycoprotein.
  4. 18 . The method according to claim 16 , wherein mammalian producer cells are incubated at step (b) in the presence of a mutagen selected from the group comprising irradiation, DNA intercalating agents, reactive oxygen species, nucleic acids, and mutagenic drugs.
  5. 19 . The method according to claim 18 , wherein the mutagen is a nucleoside analog.
  6. 20 . The method according to claim 18 , wherein the mutagen is 5-hydroxy-2′-deoxycytidine (5-OH-dC) or 5-azacytidine (5-aza-C).
  7. 21 . The method according to claim 16 , wherein mammalian producer cells are incubated at step (b) in the presence of a single-stranded or double-stranded RNA-specific deaminase or a nucleic acid coding therefor.
  8. 22 . The method according to claim 16 , wherein mammalian producer cells are incubated at step (b) in the presence of an RNA-specific adenosine deaminase or an RNA-specific cytidine deaminase.
  9. 23 . The method according to claim 21 , wherein the deaminase is fused to a programmable RNA-guided protein and the mammalian producer cells are further incubated at step (b) with a guide RNA comprising a first region at least partially complementary to a region of interest of the SoI and a second region capable of interacting with the programmable RNA-guided protein.
  10. 24 . The method according to claim 16 , wherein the pol gene expresses an error-prone reverse transcriptase/RNaseH mutant.
  11. 25 . The method according to claim 16 , wherein the pol gene expresses an error-prone reverse transcriptase/RNaseH mutant comprising at least one mutation selected from the group consisting of Y115A, M184V, M184I, Q151M, M230I and Y501W, said position number corresponding to the amino acid number of SEQ ID NO: 1.
  12. 26 . The method according to claim 16 , wherein the Retroviridae vector is a lentiviral vector.
  13. 27 . The method according to claim 16 , wherein the Retroviridae vector is a lentiviral vector selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 1 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), puma Lentivirus (PLV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), Visna-maedi virus, and Jembrana disease virus.
  14. 28 . The method according to claim 16 , wherein the Retroviridae vectors are not self-inactivating vectors.
  15. 29 . The method according to claim 16 , wherein the mammalian producer cells and/or the mammalian reporter cells are human cells.
  16. 30 . The method according to claim 16 , wherein the SoI is a coding nucleic acid sequence or a non-coding nucleic acid sequence.
  17. 31 . The method according to claim 30 , wherein the SoI is a regulatory element or expresses a non-coding RNA.
  18. 32 . The method according to claim 30 , wherein the SoI is a coding nucleic acid sequence encoding a peptide or protein.
  19. 33 . The method according to claim 16 , wherein selection of mutants of the SoI at step (f) is performed based on a measurable phenotype directly or indirectly caused by the SoI mutant in the mammalian reporter cells.
  20. 34 . The method according to claim 16 , wherein the mammalian reporter cells at steps (d) and (e) express an exogenous protein or fragment thereof acting as reporter for the selection of mutants of the SoI with a desired biological activity.

Description

FIELD OF INVENTION The present invention relates to the field of mammalian gene synthetic evolution. BACKGROUND OF INVENTION Evolution is the main architect of the existing biological world. However, protein optimization for biotherapeutic use requires tough testing of multiple variants. Scientists have coined the term “synthetic evolution” to refer to the application of modern molecular and synthetic biology approaches to iteratively diversify and select one or more targeted genetic loci with desired functions or phenotypes. This ability to accelerate evolution ultimately allows the ‘hyperdirected’ evolution of phenotypes never before seen in nature. Recreating evolution in the laboratory has been successfully used in the past to generate new protein functions with therapeutic use, such as antibodies (Jespers et al., 1994. Biotechnology (N Y). 12(9):899-903) or anti-tumor agents (Swe et al., 2012. Biochem Pharmacol. 84(6):775-783). More recently, tools and platforms for synthetic evolution have been developed (Simon et al., 2019. Nat Biotechnol. 37(7):730-743; US20030175972 A1; Rossolillo et al., 2012. PLOS Genetics; Yenerall et al., 2021. Cancer Research). In particular, virus-assisted evolution has been developed to trigger the synthetic evolution of individual genes or subset of genes. For instance, the team of David Liu at Harvard has developed “PACE”, for “phage-assisted continuous evolution”, using M13 bacteriophages containing a gene of interest to be evolved, to infect Escherichia coli cells and propagate from one cell to another. With each cycle of infection and propagation, the phage's nucleic acid including the gene of interest is subject to mutations, and phage growth enables the selection of the most active variants (Esvelt et al., 2011. Nature. 472(7344):499-503; WO 2010/028347 A2; WO 2012/088381 A2). The PACE platform, with its use of a M13 phage, is however limited to prokaryotic systems and has been mainly used for bacterial protein evolution (Badran et al., 2016. Nature. 533(7601):58-63; Bryson et al., 2018. Nat Chem Biol. 13(12):1253-1260; Hu et al., 2018. Nature. 556(7699):57-63). PACE-like methods have then been adapted to evolve mammalian genes, for instance, by coupling the replication of an adenovirus engineered to contain a mammalian gene of interest but to lack an essential protease, which is supplied in trans in mammalian cells dependently on the activity of the target gene (Berman et al., 2018. J Am Chem Soc. 140(51):18093-18103; WO 2019/084384 A1). More recently, a Sindbis RNA Alphavirus was used as vector in the “VEGAS” platform (Viral Evolution of Genetically Actuating Sequences) to evolve GPCRs and transcription factors (English et al., 2019. Cell. 178(3):748-761.e17). However, these methods and platforms are not devoid of drawbacks: first, they rely on hyper-mutagenic viruses (for instance, the Sindbis RNA Alphavirus is estimated to have a mutagenesis rate of about 10-3 to 10-4 substitutions/nucleotide/cell infection) which makes it difficult if not impossible to control mutagenesis as well as mutation spectrum (e.g., transversion/transition substitution ratio) that can occur. Indeed, given the very high mutagenesis rate of these viruses, each iteration of the method introduces not one or a few, but several mutations in the gene of interest (e.g., up to 10 mutations in a 10-kb sequence with a substitution rate of 10-3), possibly leading in a majority of cases to the co-creation of deleterious mutations and ultimately, the generation of non-functional products, which obscures the detection of beneficial mutations. It requires thus a significant amount of trials-and-error, to obtain an evolved sequence of interest with functional and advantageous mutations. Second, the viruses used in the existing platforms do not exhibit recombination events, which would however add combinatorial diversity in the process; they also do not integrate in a host cell's genome, although this may be desirable for high expression of the sequence of interest and ultimately, an easier selection of the evolved sequences of interest with a desired biological activity. Third, these methods and platforms do not allow to target a specific region of the sequence of interest to be mutated. It would however be desirable to have a method which allows to select, within a full sequence of interest, a specific region to be evolved (for instance, a region coding for a given functional domain of a gene's product). Here, we have developed a “Retroviral Synthetic Evolution” platform or “RSE” platform, the first directed evolution platform that can screen billions of protein variants in mammalian systems cost-efficiently. The platform works on the principle of iterative cycles of (i) in vivo diversification, where millions of variants of a sequence of interest are produced; (ii) in vivo encapsulation, where these variants are encoded in biological nanoparticles; and (iii) in vivo screening, where the biological nanop