Non-viral DNA vectors and uses thereof for expressing phenylalanine hydroxylase (PAH) therapeutics

Abstract

The application describes ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette encodes a transgene encoding PAH protein. Some ceDNA vectors further comprise cis-regulatory elements, including regulatory switches. Further provided herein are methods and cell lines for reliable gene expression of PAH protein in vitro, ex vivo and in vivo using the ceDNA vectors. Provided herein are method and compositions comprising ceDNA vectors useful for the expression of PAH protein in a cell, tissue or subject, and methods of treatment of diseases with said ceDNA vectors expressing PAH protein. Such PAH protein can be expressed for treating disease, e.g., Phenylketonuria (PKU).

Description

[0001] related application [0002] This application claims priority to U.S. Provisional Application No. 62 / 817,771, filed March 13, 2019, and U.S. Provisional Application No. 62 / 857,514, filed June 5, 2019, the contents of each of said provisional applications It is hereby incorporated herein by reference in its entirety. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been electronically filed in ASCII format along with the sequences in Tables 1-15 herein and each is hereby incorporated by reference in its entirety. The ASCII copy, created on March 10, 2020, is named 131698-05720_SL.txt and is 116,806 bytes in size. technical field [0005] The present invention relates to the field of gene therapy, including non-viral vectors for expressing transgenes or isolated polynucleotides in a subject or cell. The present disclosure also relates to nucleic acid constructs, promoters, vectors, and host cells, including polynucleotides, ...

AI Technical Summary

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 for heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010), so the use of AAV vectors has been limited to less than 150,000Da protein coding capacity

A second disadvantage is that rAAV gene therapy candidates must be screened for the presence of neutralizing antibodies that eliminate the vector from patients due to the prevalence of wild-type AAV infection in the population

A third disadvantage is related to capsid immunogenicity, which prevents readministration to patients not excluded from initial treatment

First, current therapies do not modify the disease, are only effective in a subset of patients, and still require strict dietary restrictions, non-adherence can lead to neuronal damage

Second, there are no approved gene therapies for PKU, and 25% to 40% of patients are ineligible for AAV-based therapies due to pre-existing antibodies

AAV can only be given once and may not produce PAH levels high enough to be effective, or may be outside the normal range and dose levels cannot be titrated

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