Figure 4.

Optimization of ZFN mRNA-LNP and AAV Transgene Donor Co-delivery for Targeted Integration of Therapeutic Transgene In Vivo

(A) Liver genome-editing results from mice injected with a single 2 mg/kg dose of LNP containing 48641/31523 ZFN mRNAs (ARCA-capped) either 1 day after (predelivery) or at the same time as (co-delivery) 1.5e12 vector genomes (vg) AAV6 or AAV8 encoding a human IDS transgene donor with homology arms flanking the ZFN cut site and a splice acceptor just upstream of the transgene coding region (n = 4 per group) (p = 0.0037 comparing AAV6 pre- and co-delivery). (B) Liver genome-editing results from mice injected with a single dose of LNP containing 48641/31523 ZFN mRNAs over the indicated dose range along with 1.5e12 vector genomes (vg) AAV8 encoding a human IDS transgene donor with homology arms flanking the ZFN cut site and a splice acceptor just upstream of the transgene coding region (p = 0.003, R² = 0.994, F = 331.7, degrees of freedom = 2). (C) IDS activity assay in plasma collected from the mice described in (B) (p = 0.0181, R² = 0.9641, F = 53.75, degrees of freedom = 2). (D) Results of liver function test (LFT) in serum collected from the mice described in (B) 1 day post-dosing. LFT, liver function test; ALT, alanine transaminase; AST, aspartate transaminase. (E) Liver genome-modification results following a single 0.5 mg/kg administration of LNP containing 25% pU substituted or unmodified 48641/31523 ZFN mRNAs co-injected with 1.5e12 vg/mouse AAV8 hIDS donor (n = 5–6 per group) (p = 0.0029). (F) IDS activity in plasma was collected from the mice described in (E) (n = 6 per group) (p = 0.011). (G) Results of liver function test (LFT) in serum collected from the mice described in (E) 1 day post-dosing. (H) Plasma IDS enzymatic activity measured from mice 28 days after co-injection with a single 1 mg/kg dose of LNP containing 48641/31523 ZFN mRNAs, purified either via silica column or HPLC and 1.5e12 vg/mouse AAV8 hIDS donor (n = 3–4 per group) (p = 0.055). (I) Results of liver function test (LFT) in serum collected from the mice described in (H) 1 day post-dosing (n = 4 per group) (p = 0.0015 comparing silica versus HPLC ALT; p = 0.0095 comparing AST). (J) Liver genome-editing activity results following single doses of LNP containing 48641/31523 ZFN mRNAs (enzymatic Cap1) injected into mice at 2 mg/kg at the same time as 1.5e12 vector genomes (vg) AAV8 encoding a human IDS transgene donor. Animals were either pretreated with dexamethasone just prior to LNP dosing or just prior to and for an additional 3 days after dosing. (K) IDS activity in plasma was collected from the mice described in (J). (L) Liver genome-editing results following single doses of LNP containing 48641/31523 ZFN mRNAs injected into mice at 2 or 3.5 mg/kg at the same time as 1.5e12 vector genomes (vg) AAV8 encoding a human FIX transgene donor. The FIX donor comprised homology arms flanking the ZFN cut site and a splice acceptor just upstream of the transgene coding region (n = 3–4 per group) (p = 0.047). (M) FIX protein expression results in plasma collected from the mice described in (L).