Co-packaging to mitigate intermolecular recombination

Abstract

The subject matter disclosed herein is generally directed to methods and compositions for stable transduction of target cells with libraries of genetic elements. The invention reduces intermolecular recombination between library elements and integration of multiple genetic elements.

1 - 37 . (canceled) 
     
     
         38 . A non-naturally occurring engineered lentiviral or retroviral system comprising:
 a) a plurality of payload vectors, each encoding a lentiviral or retroviral genome comprising at least one genetic perturbation and a barcode sequence that identifies each genetic perturbation, wherein each genome sequence is modified to reduce dimerization of the genomes; and   b) one or more packaging vectors encoding lentiviral or retroviral packaging proteins for generating packaging cells or a packaging cell line encoding the lentiviral or retroviral packaging proteins,
 whereby transfection of the plurality of payload vectors into packaging cells produces a viral expression library comprising viral particles biased to a single genome per virion. 
   
     
     
         39 . The engineered system of  claim 38 , wherein the reduced dimerization comprises deletion, introduction, and/or relocation of a dimer initiation site/dimer linkage sequence (DIS/DLS) region in the genome sequence. 
     
     
         40 . The engineered system of  claim 38 , wherein the reduced dimerization comprises reduced hairpin formation. 
     
     
         41 . The engineered system of  claim 40 , wherein stem-loop 1 (SL1) in the DIS/DLS is modified or knocked out. 
     
     
         42 . The engineered system of any of  claim 38 , wherein the genome sequence is modified to reduce or knock out U5:AUG pairing. 
     
     
         43 . A non-naturally occurring engineered lentiviral or retroviral system comprising:
 a) a plurality of payload vectors, each encoding a lentiviral or retroviral genome comprising at least one genetic perturbation and a barcode sequence that identifies each genetic perturbation; and   b) one or more packaging vectors encoding lentiviral or retroviral packaging proteins for generating packaging cells or a packaging cell line encoding the lentiviral or retroviral packaging proteins, wherein the packaging proteins comprise one or more modified packaging proteins capable of reducing recombination, template switching, and/or multiple integration of the lentiviral or retroviral genomes in transduced viral particles, optionally, further comprising an inhibitor of template switching,   whereby transfection of the plurality of payload vectors in the packaging cells produces a viral expression library comprising viral particles, such that target cells transduced with the viral expression library have reduced recombination, template switching, and/or multiple integration of the lentiviral or retroviral genomes.   
     
     
         44 . The engineered system of  claim 43 , wherein a packaging protein involved in dimerization is modified, knocked down, or knocked out. 
     
     
         45 . The engineered system of  claim 44 , wherein a nucleocapsid (NC) protein is modified, knocked down, or knocked out. 
     
     
         46 . The engineered system of  claim 45 , wherein the nucleocapsid (NC) protein is modified by a mutation in the zinc-finger region of the NC. 
     
     
         47 . The engineered system of  claim 43 , wherein template-switching activity of the reverse transcriptase is knocked down or knocked out. 
     
     
         48 . The engineered system of  claim 43 , wherein gag polyprotein cleavage is inhibited by a mutation in a protease. 
     
     
         49 . A non-naturally occurring engineered lentiviral or retroviral system comprising:
 a) a plurality of payload vectors, each encoding a lentiviral or retroviral genome comprising at least one genetic perturbation and a barcode sequence that identifies each genetic perturbation;   b) a carrier vector encoding for a polynucleotide designed to hybridize with all or part of the 5′ UTR of the lentiviral or retroviral genome; and   c) one or more vectors encoding lentiviral or retroviral packaging proteins for obtaining packaging cells or a packaging cell line encoding lentiviral or retroviral packaging proteins,
 whereby co-transfection of the plurality of payload vectors and the carrier vector into packaging cells produces a viral expression library comprising viral particles biased to a single genome per virion. 
   
     
     
         50 . The engineered system of  claim 49 , wherein the polynucleotide is designed to hybridize with the U5-PBS complex. 
     
     
         51 . The engineered system of  claim 49 , wherein the polynucleotide is designed to hybridize with the dimer initiation site (DIS). 
     
     
         52 . A non-naturally occurring engineered lentiviral or retroviral system comprising:
 a) a plurality of payload vectors, each encoding a lentiviral or retroviral genome comprising at least one genetic perturbation and a barcode sequence that identifies each genetic perturbation;   b) a plurality of carrier vectors, wherein the plurality of carrier vectors:
 i. encode for a non-recombinogenic lentiviral or retroviral genome capable of being packaged into a viral particle as a dimer with a lentiviral or retroviral genome encoded for by the payload vector and not capable of recombination with the lentiviral or retroviral genome encoded for by the payload vector; or 
 ii. does not encode for a lentiviral or retroviral genome and is not capable of being packaged into a viral particle; and 
   c) one or more vectors encoding lentiviral or retroviral packaging proteins for obtaining packaging cells or a packaging cell line encoding lentiviral or retroviral packaging proteins,
 whereby co-transfection of the plurality of payload vectors and the carrier vectors into packaging cells produces a viral expression library comprising viral particles, such that target cells transduced with the viral expression library have reduced recombination, template switching, and/or multiple integration of the lentiviral or retroviral genomes as compared to packaging cells transfected with only the plurality of payload vectors. 
   
     
     
         53 . The engineered system of  claim 52 , wherein the non-recombinogenic lentiviral or retroviral genome is a non-integrating lentivirus genome. 
     
     
         54 . The engineered system of  claim 52 , wherein the ratio of carrier vector to payload vector is greater than 5:1. 
     
     
         55 . The engineered system of any of  claim 38 , further comprising an inhibitor of template switching. 
     
     
         56 . The engineered system of any of  claim 38 , wherein the genetic perturbation comprises an over expressed gene, RNAi based system, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a meganuclease, a CRISPR-Cas system, or a component thereof. 
     
     
         57 . The engineered system of  claim 56 , wherein the genetic perturbation comprises a CRISPR-Cas system or a component thereof. 
     
     
         58 . The engineered system of  claim 57 , wherein the CRISPR-Cas system is a CRISPR-Cas9 system. 
     
     
         59 . The engineered system of  claim 58 , wherein the genetic perturbation is one or more guide sequences. 
     
     
         60 . A method of screening cells for at least one genetic perturbation comprising:
 providing a population of cells;   introducing a viral expression library obtained using the system of  claim 38  to the population of cells, such that each cell receives a lentiviral or retroviral genome encoded for by a payload vector;   detecting genomic, genetic, proteomic, epigenetic and/or phenotypic differences in single cells receiving a lentiviral or retroviral genome; and   identifying the genetic perturbation in each cell based on the barcode sequence.   
     
     
         61 . A viral expression library comprising viral particles obtained using the system of  claim 38 . 
     
     
         62 . A packaging cell line for producing lentiviral or retroviral viral particles comprising:
 a) polynucleotide sequences encoding lentiviral or retroviral packaging proteins, wherein the packaging proteins comprise one or more modified packaging proteins capable of reducing recombination, template switching, and/or multiple integration of the lentiviral or retroviral genomes in transduced viral particles, optionally, further comprising an inhibitor of template switching; or   b) polynucleotide sequences encoding lentiviral or retroviral packaging proteins, wherein the packaging proteins comprise an inhibitor of template switching.   
     
     
         63 . The packaging cell line of  claim 62 , wherein a packaging protein involved in dimerization is modified, knocked down, or knocked out. 
     
     
         64 . The packaging cell line of  claim 63 , wherein a nucleocapsid (NC) protein is modified, knocked down, or knocked out. 
     
     
         65 . The packaging cell line of  claim 64 , wherein the nucleocapsid (NC) protein is modified by a mutation in the zinc-finger region of the NC. 
     
     
         66 . The packaging cell line of  claim 62 , wherein template-switching activity of the reverse transcriptase is knocked down or knocked out. 
     
     
         67 . The packaging cell line of  claim 62 , wherein gag polyprotein cleavage is inhibited by a mutation in a protease. 
     
     
         68 . A method of reducing intermolecular recombination between lentiviral genomes in a transduced library comprising packaging viral particles for transduction using the system of  claim 38 .