Methods and compositions for modification of a cystic fibrosis transmembrane conductance regulator (CFTR) gene
A composition and gene technology, applied in the field of genome editing, can solve the problems of undetectable long-term expression of CFTR, obstruction, and inability to achieve stable transduction of stem/progenitor cell populations
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Embodiment 1
[0219] Example 1: Materials and methods
[0220] ZFN
[0221] ZFNs targeting introns 1, 2, 3, 6, 7, and 8 of the murine or human CFTR gene were designed and screened in Neuro2a and K562 cells, respectively. A ZFN targeting the ΔF508 mutation within exon 11 of the human CFTR gene was also designed and screened in a K562 cell line engineered to contain 1 of 5 alleles with the corresponding ΔF508 mutation alleles, as well as the four wild-type alleles, and are shown in Tables 1A and 1B. See also US Patent No. 9,161,995 for SBS #32401 in Table IB. Target sites are shown in Table 2. Nucleotides in the target site targeted (contacted) by the ZFP recognition helix are indicated in upper case; non-targeted (contacted) nucleotides are indicated in lower case.
[0222] ZFNs are formed in a ZFP-Fok or Fok-ZFP orientation (US Patent No. 7,972,854 and US Patent Publication No. 2017 / 0218349), paired with obligate heterodimers such as ELD and KKR. See, eg, US Patent Nos. 7,914,796; 8,...
Embodiment 2
[0240] Example 2: Genetic Modifications within the CFTR Intron
[0241] a. NHEJ modification (indel%)
[0242] Nucleases targeting CFTR introns 1, 2, 3, 6, 7 or 8 were assayed as described in Example 1 (Table 1A above). Additionally, the activity of nucleases targeting the ΔF508 mutation was also assayed as described above and in US Pat. No. 9,161,995.
[0243] Specifically, for human CFTR, 113 ZFN pairs targeting introns 6–8 and 95 ZFN pairs targeting introns 1–3 were tested and superior candidates were identified. ZFNs were electroporated as plasmid DNA into K562 cells in an Amaxa facility and harvested after 3 days for indel analysis by Miseq.
[0244] Such as figure 1 As shown, the superior candidates identified against human CFTR are: against intron 1 (56316 / 56317-52% indel), against intron 3 (56445 / 56444-32.7% indel), against intron 7 (56126 / 56127-55.4% indel), and against intron 8 (56255 / 56254-50.1% indel).
[0245] Similarly, for mouse CFTR, 48 ZFN pairs targe...
Embodiment 3
[0263] Example 3: Ex vivo and in vivo methods
[0264] Patient-derived basal epithelial lung cells, lung stem cells, or iPSCs are modified as described in Example 2, for example, are administered to a subject with CF in the form of an inhalable formulation, and the transplanted cells are produced in the subject Therapeutic levels of protein.
[0265] Alternatively, the nuclease (eg, intron-targeted) and the CFTR donor are administered to the CF patient, eg, via an inhalable formulation and / or using a vector (eg, AAV specifically targeted to lung cells). The subject is treated by incorporating the CFTR donor into lung cells of the subject using the same or a different delivery vehicle and producing functional CFTR in the lung cells.
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