(643) Delivery of gp64-pseudotyped lentivirus carrying codon-optimized CFTR provides improved functional restoration in human CF airway epithelial cultures

Thursday, November 3, 2022

12:45 PM – 1:25 PM ET

Presenting Author(s)

MM

Madhu Mahankali, PhD (she/her/hers)

Principal Scientist
Spirovant Sciences, Inc
Wayne , Pennsylvania, United States

Co-Author(s)

MJ

Mohamad Jamiluddin

United States
HG

Hassan Gheisari

United States
MW

Matthew Weaver

United States
AR

Aniqua Rahman-Zaman

United States
CU

Cindy Ung

United States
RK

Roland Kolbeck

Chief Scientific Officer
Spirovant Sciences, Inc, United States
Katherine Excoffon, PhD (she/her/hers)

VP of Research
Spirovant Sciences, Inc
Philadelphia, Pennsylvania, United States
JD

Julian Down

United States

Background: Cystic fibrosis (CF) is caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR) protein, resulting in impaired chloride conductance across epithelia lining the respiratory airways. Gene therapy using lentiviral vectors (LVs) offers a promising approach for treating CF through stable genome integration for long-term CFTR transgene expression. Pseudotyping lentiviruses with specific glycoproteins improves their infectivity and modifies tropism. LVs pseudotyped with gp64, a baculovirus envelope protein, have previously been shown to effectively transduce airway epithelia to deliver the CFTR gene in a pig model of cystic fibrosis [1]. We have developed gp64-pseudotyped LV vectors carrying the coding sequence for human CFTR (gp64-LV-hCFTR), insulator elements for improved safety and expression, and a promoter that constitutively drives expression of the gene for hCFTR. Marquez Loza et al [2] utilized codon-optimized CFTR constructs and demonstrated significant increase in CFTR function. Following that precedence, we further designed various codon-optimized versions of gp64-LV-hCFTR to minimize long term gene silencing and for the establishment of codon-specific molecular assays for hCFTR detection, quantification, and localization of mRNA transcripts.

Methods: Codon-optimized versions of gp64-LV-hCFTR were designed and the LV constructs were evaluated in fisher rat thyroid (FRT) cells and primary CF human airway epithelia (HAE). CF-HAE or FRT cells were transduced with gp64-LV vectors at the time of seeding at different multiplicity of infection (MOI) and cells were allowed to differentiate at the air liquid interface (ALI). CFTR-specific chloride conductance was measured by Ussing chamber assay. Vector copy number (VCN) was determined by droplet digital PCR (ddPCR) assay.

Results: At 21 days post-transduction, gp64-LV-hCFTR restored CFTR-mediated chloride conductance across polarized HAE layers in a MOI dose dependent manner. Correction of CF varied depending on the lentiviral purification process and correlated with increasing VCN with full functional restoration achieved at ≤5 VCN/cell. Using a gp64-LV reporter vector containing identical regulatory elements we were also able to demonstrate reporter gene expression more than 9 months in transduced CF HAE cultures, indicating durability of transgene expression. Assessment of codon-optimized CFTR plasmids in FRT cells showed further improvement of CFTR correction over wildtype hCFTR. The codon-optimized CFTR LV constructs also showed successful transduction and improved CFTR functional activity by 2 to 3-fold while maintaining the VCN similar to wildtype CFTR in four different primary CF-HAE donors tested.

Conclusions: In summary these data suggest the potential for improved therapeutic efficacy of gp64-LV-hCFTR with a codon-optimized hCFTR transgene. This approach not only improves CFTR correction significantly when compared to wildtype CFTR but also allows the design of specific and sensitive molecular assays for detection, quantification, and localization of the vector and gene expression.

Acknowledgements:

References:

[1] Cooney et al. JCI Insight. 2016;1(14):e88730.

[2] Marquez Loza et al. Mol Ther Methods Clin Dev. 2021;21:94-106.