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EP-4739782-A2 - VECTORS AND METHODS FOR BUILDING POLYCISTRONIC AND/OR MULTIGENIC TRANSGENES

EP4739782A2EP 4739782 A2EP4739782 A2EP 4739782A2EP-4739782-A2

Abstract

Cloning vectors and methods useful for building multigenic and/or polycistronic constructs for delivery to cells and/or organisms are disclosed. The vectors are genetically engineered to comprise unique acceptor domains that independently enable the sequential or iterative addition of compliant DNA and/or RNA inserts. Insertion of a first nucleotide insert into an acceptor r domain destroys a complementary restriction site a 3'end of the first insert segment while the 3' restriction site is regenerated at the insertion point of the cloning vector to form a circular vector with a first insert. A subsequent cleavage at a 3' end of the first insert creates an insertion point for a second insert segment, and the iterative assembly of additional insert segments proceeds with each subsequent cleavage at a 3' end of a growing linear chain of a desired number of inserts.

Inventors

  • REED, THOMAS
  • SCHAUER, Stephen
  • PEPPEL, Austin

Assignees

  • Biosolutions Designs

Dates

Publication Date
20260513
Application Date
20240703

Claims (20)

  1. 1. A method for building polycistronic or multigenic transgenes, comprising linearizing a first plasmid DNA vector to create a first acceptor site comprising a first restriction site with a 5’ end and a 3’ end, providing a first nucleotide fragment has a 5’ end and a 3’ end that are complimentary to the 5 ’end and the 3’ end of the first restriction site; inserting the first nucleotide fragment into the first acceptor site, ligating the first insertion nucleotide fragment ends to the acceptor site ends, wherein ligating regenerates the 3’ end of the first restriction site, and destroys the 5’ end of the first restriction site while generating a further 5’ end of the first restriction site; and performing the linearizing, inserting and ligating steps with one or more additional nucleotide fragments, wherein each of the one or more additional nucleotide fragments is different from the first nucleotide fragments, and producing a multigenic and/or polycistronic transgene comprising the first insertion segment and the one or more additional DNA segments linked end to end in a linear chain.
  2. 2. The method of claim 1, wherein the selected plasmid DNA vector has a nucleotide sequence identity selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
  3. 3. The method of claim 1, further comprising: providing a set of interrelated plasmid DNA vectors genetically engineered to have one or more unique acceptor sites and a plurality of compliant genetically engineered nucleotide fragments wherein each compliant genetically engineered fragment has 5’ and 3’ ends that are compatible with one of the one or more unique acceptor sites, wherein said first plasmid DNA vector is one vector in the set, and excising the multigenic and/or polycistronic transgene from the first plasmid DNA vector and inserting the first plasmid DNA vector into a second plasmid DNA vector of said set.
  4. 4. A plasmid DNA cloning vector, comprising one or more genetically engineered unique acceptor sites, wherein the vector is configured for sequentially and/or iteratively producing a multigenic and/or polycistronic construct.
  5. 5. The plasmid DNA cloning vector of claim 4, wherein the plasmid DNA cloning vector has a nucleotide sequence identity selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
  6. 6. The plasmid DNA cloning vector of claim 5, wherein the nucleotide sequence identity is SEQ ID NO:1.
  7. 7. The plasmid DNA cloning vector of claim 5, wherein the nucleotide sequence identity is SEQ ID NO:2.
  8. 8. The plasmid DNA cloning vector of claim 5, wherein the nucleotide sequence identity is SEQ ID NO:3.
  9. 9. The plasmid DNA cloning vector of claim 5, wherein the nucleotide sequence identity is SEQ ID NO:4.
  10. 10. The plasmid DNA cloning vector of claim 5, wherein the nucleotide sequence identity is SEQ ID NO:5.
  11. 11. The plasmid DNA cloning vector of claim 5, wherein the nucleotide sequence identity is SEQ ID NO:6.
  12. 12. The plasmid DNA cloning vector of claim 5, wherein the nucleotide sequence identity is SEQ ID NO:7.
  13. 13. A combination of nucleotide molecules for assembly of multigenic and/or polycistronic constructs, comprising: a plasmid DNA cloning vector genetically engineered to have one or more unique acceptor sites; and one or more compliant genetically engineered fragments which are insertable into at least one of the one or more unique acceptor sites, wherein each compliant genetically engineered fragment has 5’ and 3’ ends which are compatible with a single one of the one or more unique acceptor sites, and wherein the plasmid DNA cloning vector and each of the one or more compliant genetically engineered fragments are designed such that ligation of one of the one or more genetically engineered fragments into the single one of the one or more unique acceptor sites destroys a 5’ end of the acceptor site and regenerates a 3’ end of the acceptor site.
  14. 14. The combination of claim 13, wherein the one or more unique acceptor sites comprises a plurality of acceptor sites, and wherein the plasmid DNA cloning vector further comprises a plurality of spacers with a spacer between each of the acceptor sites of the plurality of acceptor sites, with the caveat that each spacer is different from every other spacer in the plasmid DNA cloning vector.
  15. 15. The combination of claim 13, wherein the plurality of spacers is selected from the group consisting of CAGAGTCCC, GGGAGGTTT, ACCTCAAGG, GCAGAAGTC, AGCCAACCT, TGCCGAGTC, CCAGCCGCC, GAAGAGGT, CACTTCCTG, CTCTGAGCC, AGCCTCAGT and ATATCACGC.
  16. 16. The combination of claim 13, wherein each of the one or more compliant genetically engineered fragments is genetically engineered to not have coding for Sall, Pvul, KasI, Agel, Xhol, Notl, and Asel restriction enzyme recognition sites.
  17. 17. The combination of claim 16, wherein each of the one or more compliant genetically engineered fragments is further genetically engineered to not have coding for Seal restriction enzyme recognition sites.
  18. 18. The combination of claim 16, wherein each of the one or more compliant genetically engineered fragments is further genetically engineered to not have coding for Bsgl restriction enzyme recognition sites.
  19. 19. A combination of nucleotide molecules and nucleotide molecule segments for assembly of multigenic and/or polycistronic constructs, comprising: a first nucleotide molecule genetically engineered to have one or more unique acceptor sites; and one or more compliant genetically engineered nucleotide molecule segments which are insertable into at least one of the one or more unique acceptor sites, wherein each compliant genetically engineered nucleotide molecule segment has 5’ and 3’ ends that are compatible with a single one of the one or more unique acceptor sites, and wherein the first nucleotide molecule and each of the one or more compliant genetically engineered nucleotide molecule segments are designed such that ligation of one of the one or more genetically engineered nucleotide molecule segments into the single one of the one or more unique acceptor sites destroys a 5’ end of the acceptor site and regenerates a 3’ end of the acceptor site.
  20. 20. The combination of claim 19, wherein the one or more unique acceptor sites comprises a plurality of acceptor sites, and wherein the first nucleotide molecule further comprises a plurality of spacers with a spacer between each of the acceptor sites of the plurality of acceptor sites, with the caveat that each spacer is different from every other spacer in the first nucleotide molecule.

Description

VECTORS AND METHODS FOR BUILDING POLYCISTRONIC AND/OR MULTIGENIC TRANSGENES CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63/511,725, filed July 3, 2023, which is hereby incorporated herein by reference in its entirety. SEQUENCE LISTING This application includes as the Sequence Listing the complete contents of the accompanying text file “Sequence.txt", created July 1, 2024, containing 25000 bytes, hereby incorporated by reference. BACKGROUND There are many types of cloning vectors, but the most commonly used ones are genetically engineered plasmids. A plasmid is a naturally occurring small circular piece of DNA which can be stably maintained in an organism such as bacteria. Plasmids have been engineered as useful tools into which a foreign DNA fragment can be inserted for cloning purposes. Commercial vectors contain features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, such as restriction enzyme recognition sites. The vector and a fragment of exogenous DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments, generating either blunt ends or overhangs known as sticky ends. Vector DNA and DNA insert(s) with compatible ends can then be joined by molecular ligation. Typically, a series of unique restriction sites are clustered within a multiple cloning site or polylinker. After a DNA fragment has been ligated into a cloning vector, it may be further subcloned into another vector designed for a more specific use. All commonly used cloning vectors in molecular biology have key features necessary for their function, such as a selectable marker. Others may have additional features specific to their use. For reason of ease and convenience, cloning is often performed using E. coli. Thus, the cloning vectors used often have elements necessary for their propagation and maintenance in E. coli, such as a functional origin of replication (ori). The ColEl origin of replication is found in many plasmids. Some vectors also include elements that allow them to be maintained in another organism in addition to E. coli. With the advent of gene therapy, the scientific community has recognized a need for delivery of multiple genes for treatment of complex diseases. It would be advantageous to deliver a multigene therapy to all target cells that allows expression of all the genes of interest in those cells. Delivery of multiple types of vectors can result in uneven uptake into target cells, resulting in uneven distribution of transgene expression. Many legacy or prior art vectors, including those taught in US 10036026 and US20080050808, were designed for use when de novo synthesis of DNA was unreliable and cloning of genomic fragments or cDNA fragments was considered the best practice to ensure fidelity of the nucleotide sequence. Despite the widespread commercialization and uses of plasmid cloning vectors, there are no specialized vectors engineered to allow rapid and iterative or sequential construction of multigenic and/or polycistronic constructs within a single vector. The larger the construct becomes, the more likely it is that duplications of restriction sites in the multiple cloning site and the DNA inserts make it difficult, if not impossible, to assemble and excise a multigenic and/or polycistronic construct. Thus, a need continues to exist for a vector that has the capability to deliver multiple gene products within a single payload. Additionally, there is a need for a standardization of genetic modules so that useful genetic components can be linked together in a logical fashion and be swapped between multiple classes of plasmid DNA vectors. More specifically, there is a need to develop a standardized approach for building monogenic vectors and multigenic vectors useful for in vitro transcription, transient transfection, and stable integration. SUMMARY OF THE INVENTION An aspect of the invention relates to a method for building polycistronic or multigenic transgenes, comprising providing a set of interrelated plasmid DNA vectors genetically engineered to have one or more unique acceptor sites and a plurality of compliant genetically engineered nucleotide fragments wherein each compliant genetically engineered fragment has 5’ and 3’ ends that are compatible with one of the one or more unique acceptor sites. A first plasmid DNA vector is selected and linearized at unique restriction site. This creates a first acceptor site comprising a first restriction site with a 5’ end and a 3’ end, wherein a first nucleotide fragment has a 5’ end and a 3’ end that arc complimentary to the 5 ’end and the 3’ end of the first restriction site. The first nucleotide fragment is inserted into the first acceptor site, And ligating the first insertion nucleotide fragment ends to the acceptor site ends regenerates the 3’ end of the first restriction site and destroys the 5’ end of the first