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Modified closed-ended DNA (CEDNA) comprising symmetrical modified inverted terminal repeats

A closed-end, symmetric technology that can be used in the field of gene therapy to solve problems such as limited viral packaging capacity

Pending Publication Date: 2021-08-27
GENERATION BIO CO
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009]However, there are several major drawbacks to the use of AAV particles as gene delivery vehicles
A major disadvantage associated with rAAV is its limited viral packaging capacity of approximately 4.5 kb of heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010)
As a result, the use of AAV vectors has been limited to less than 150,000 Da of protein-coding capacity
A second disadvantage is that, due to the prevalence of wild-type AAV infection in the population, rAAV gene therapy candidates must be screened for the presence of neutralizing antibodies that eliminate the vector from patients
A third disadvantage is related to capsid immunogenicity, which prevents readministration to patients not excluded from initial treatment
However, constant expression of transgenes may not be desirable in all cases

Method used

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  • Modified closed-ended DNA (CEDNA) comprising symmetrical modified inverted terminal repeats
  • Modified closed-ended DNA (CEDNA) comprising symmetrical modified inverted terminal repeats
  • Modified closed-ended DNA (CEDNA) comprising symmetrical modified inverted terminal repeats

Examples

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example

[0564] The following examples are offered by way of illustration and not limitation.

example 1

[0565] Example 1: Construction of ceDNA vector

[0566] The use of polynucleotide construct templates to generate ceDNA vectors is described in Example 1 of PCT / US18 / 49996. For example, the polynucleotide construct template used to generate the ceDNA vectors of the invention can be a ceDNA-plasmid, ceDNA-bacmid and / or ceDNA-baculovirus. Without being bound by theory, in a permissive host cell, in the presence of, for example, Rep, a polynucleotide construct template having two symmetrical ITRs and an expression construct is replicated to generate a ceDNA vector, wherein at least one of the ITRs is relative to The wild-type ITR sequence is modified. ceDNA vector generation goes through two steps: first, the template is cleaved ("rescued") from the template backbone (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus genome, etc.) by the Rep protein; and second, the cleavage The ceDNA vector under the Rep-mediated replication.

[0567] One exemplary method of generating a c...

example 2

[0595] Example 2: Generation of synthetic ceDNA from double-stranded DNA molecules by excision

[0596] Synthetic production of ceDNA vectors is described in Examples 2-6 of International Application PCT / US19 / 14122, filed January 18, 2019, which is incorporated herein by reference in its entirety. One exemplary method of producing ceDNA vectors using synthetic methods involves excision of double-stranded DNA molecules. Briefly, ceDNA vectors can be generated using double-stranded DNA constructs, see e.g. PCT / US19 / 14122 Figure 7A -8E. In some embodiments, the double-stranded DNA construct is a ceDNA plasmid, see, eg, Figure 6 of International Patent Application PCT / US2018 / 064242 filed December 6, 2018).

[0597] In some embodiments, a construct for making a ceDNA vector comprises a regulatory switch as described herein.

[0598] For purposes of illustration, Example 1 describes the generation of a ceDNA vector, an exemplary closed DNA vector generated using this method. Ho...

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Abstract

Described herein are ceDNA vectors having linear and continuous structure can be produced in high yields and used for effective transfer and expression of a transgene. According to some embodiments, ceDNA vectors comprise at least one heterologous nucleotide sequence operably positioned between two flanking symmetric inverted terminal repeat sequences that are not wild-type AAV ITR, wherein all or part of the heterologous nucleotide sequence is under the control of at least one regulatory switch. Some ceDNA vectors provided herein further comprise cis-regulatory elements and provide high gene expression efficiencies. Further provided herein are methods and cell lines for reliable and efficient production of the linear, continuous and capsid-free DNA vectors.

Description

[0001] related application [0002] This application claims priority to U.S. Provisional Application No. 62 / 757,872, filed November 9, 2018, and U.S. Provisional Application No. 62 / 757,892, filed November 9, 2018, the contents of each of said provisional applications It is hereby incorporated herein by reference in its entirety. technical field [0003] The present invention relates to the field of gene therapy involving the delivery of closed DNA regulatory switches to target cells, tissues, organs or organisms. [0004] sequence listing [0005] The sequence listing of this application has been electronically submitted as a sequence listing in ASCII format through EFS-Web, the file name is "05320Sequence_Listing.txt", the creation date is November 8, 2019 and the size is 204KB (209,626 bytes). This application contains a Sequence Listing, which was filed electronically and is hereby incorporated by reference in its entirety. Background technique [0006] Gene therapy...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/85C12N15/86
CPCC12N15/85C12N2750/14122C12N2750/14143A61K48/0091C12N15/86A61K2039/505A61K48/00C12N2800/107
Inventor R·M·科廷O·阿尔坎A·琼斯
Owner GENERATION BIO CO
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