(I-325) Evaluation of engineered promoters for homology-independent targeted integration of curative promoter-driven transgenes for hemophilia therapeutics

Hemophilia B is a congenital, X-linked genetic disease that creates a deficiency or absence of coagulation factor IX (FIX). This deficiency increases the chance of spontaneous and prolonged bleeding due to the body’s inability to create clots, leading to severe complications¹. An estimated 30,000 men suffer from hemophilia in the U.S., with around 6,000 cases being hemophilia B. Over 40% of patients with hemophilia produce less than 1% of normal coagulation factor levels². Infusions of coagulation factor concentrates are the most common prophylactic treatment for hemophilia B patients¹, administered anywhere from every other day to weekly³. Many researchers have looked to gene therapy to mitigate the treatment burden, including Hemgenix, the first hemophilia B gene therapy treatment approved by the FDA⁴.

The liver is the most frequently targeted organ for FIX therapy. However, skeletal muscle can be utilized as a regenerative protein factory to produce therapeutic levels of FIX. The skeletal muscle accounts for over 30% of the body’s mass⁵, indicating the possibility of large protein production. This project will optimize non-endogenous protein production in skeletal muscle, such as FIX production. Proteins needed for FIX processing export are expressed in muscle but at a lower rate than in the liver⁶. This indicates that reaching therapeutic levels of muscle-synthesized FIX will require additional expression elements. This study examines three promoters for FIX expression in muscle: a ubiquitous promoter from cytomegalovirus (CMV), a muscle-specific promoter from creatine kinase (CK8e), and an additional synthetic muscle-specific promoter (SPc5-12).

Plasmids containing the human FIX coding sequence were excised, cloned into plasmids containing CMV and CK8 promoters, and transformed into BL21(DE3)pLysS and DH5α E. Coli cells. DNA midiprep was used after full-length sequence verification to purify the plasmid. Human epithelial cells (HEK293) and mouse myoblast cells (C2C12) were transfected with the isolated plasmid using three different methods: Lipofectamine 3000, electroporation, and TransIT®-LT1, following the manufacturer’s protocol. The myoblast cells were differentiated once full confluency was reached and grown in low serum media for another week. The cell media was collected and replaced for all samples every three days, including before the differentiation of the C2C12 cells. The cell media was then analyzed using enzyme-linked immunoassay (ELISA) and western blot to measure the quantity of produced FIX and immunostaining to confirm protein identity. The cells were harvested for RNA and qRT-PCR was used to measure human FIX expression under different promoters. A commercially produced pure human FIX protein acted as a control for the study.

Based on qRT-PCR, we found that the gene expression of human FIX in myotubes under the CK8 promoter is the same as the CMV promoter after normalization with peptidylprolyl isomerase A (PPIA), a housekeeping gene used as a reference. CMV is a strong, ubiquitous promoter. However, CK8 and SPc5-12 are muscle-specific and indicated in many studies as stronger promoters in skeletal muscle^7,8. The ubiquitous nature of CMV poses increased risks of immunogenicity and insertional mutagenesis. Semi-random integration of constructs containing the CMV promoter could lead to the activation of oncogenes as previously shown in animal models, though this has not been observed in human clinical trials. SPc5-12 and CK8 are inactive in non-muscle tissues, making them a safer option for therapy using viral vectors⁷. These preliminary results will be further developed and implemented in mouse studies. Human FIX under different promoters will be delivered using adeno-associated virus (AAV) vectors targeted at skeletal muscle. The protein expression will be measured to elucidate the gene expression capacity under a promoter.

The continued study will also show the ability of skeletal muscle to make post-translational modifications to non-endogenous protein. Mature FIX features many post-translational modifications, with some before protein secretion. The most notable modifications are propeptide cleavage by Furin and γ-carboxylation by γ-glutamyl carboxylase⁶. Both proteins are expressed ubiquitously but display higher expression in the liver^9,10.  Achieving therapeutic levels of FIX, a non-ubiquitous protein, could be applied to other gene therapies aiming to deliver therapeutic proteins to the muscle. In addition to optimizing the use of promoters in muscle-targeted gene delivery, we are examining the use of hyperactive proteins to further increase protein production efficiency in muscle. Mutations PADUA (R338L), which increases FIX activity around 8-fold, and K5A, which increases circulating FIX levels around 2-fold, have been shown to increase the efficiency of hemophilia B gene therapies¹¹. The completion of this project will assist in developing an efficient hemophilia B treatment with simple administration and other gene therapies targeting muscle protein production.

1. National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, U.S. Department of Health & Human Services. 2023. Hemophilia. https://www.cdc.gov/ncbddd/hemophilia/index.html

2. Soucie, J. M., Evatt, B., & Jackson, D. (1998). Occurrence of hemophilia in the United States. The Hemophilia Surveillance System Project Investigators. American journal of hematology, 59(4), 288–294. https://doi.org/10.1002/(sici)1096-8652(199812)59:4< 288::aid-ajh4 >3.0.co;2-i

3. Horava, S.D., & Peppas, N.A. (2017). Recent advances in hemophilia B therapy. Drug Deliv. and Transl. Res. 7, 359–371. https://doi.org/10.1007/s13346-017-0365-8

4. U.S. Food and Drug Administration. 2022. FDA Approves First Gene Therapy to Treat Adults with Hemophilia B. https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapy-treat-adults-hemophilia-b

5.  Janssen, I., Heymsfield, S. B., Wang, Z., & Ross, R. (2000). Skeletal muscle mass and distribution in 468 men and women aged 18–88 yr. Journal of Applied Physiology. https://doi.org/10.1152/jappl.2000.89.1.81

6.  Zacchi, L.F., Roche-Recinos, D., Pegg, C.L., Phung, T. K., Napoli, M., Aitken, C., Sandford, V., Mahler, S. M., Lee, Y. Y., Schulz, B. L., & Howard, C. B. (2021). Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product. Commun Biol, 4, 390. https://doi.org/10.1038/s42003-021-01903-x

7. Skopenkova, V. V., Egorova, T. V., & Bardina, M. V. (2021). Muscle-Specific Promoters for Gene Therapy. Acta naturae, 13(1), 47–58. https://doi.org/10.32607/actanaturae.11063

8. Tabebordbar, M., Lagerborg, K. A., Stanton, A., King, E. M., Ye, S., Tellez, L., Krunnfusz, A., Tavakoli, S., Widrick, J. J., Messemer, K. A., Troiano, E. C., Moghadaszadeh, B., Peacker, B. L., Leacock, K. A., Horwitz, N., Beggs, A. H., Wagers, A. J., & Sabeti, P. C. (2021). Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell, 184(19), 4919–4938.e22. https://doi.org/10.1016/j.cell.2021.08.028

9. Abdel-Majid Khatib ; Fatma Sfaxi. FURIN (furin (paired basic amino acid cleaving enzyme)). Atlas Genet Cytogenet Oncol Haematol. 2012-04-01. Online version: https://atlasgeneticsoncology.org/gene/40646/furin-(furin-(paired-basic-amino-acid-cleaving-enzyme))

10.  Wu, S., Stafford, D. W., Frazier, L. D., Fu, Y., High, K. A., Chu, K., Sanchez-Vega, B., & Solera, J. (1997). Genomic Sequence and Transcription Start Site for the Human γ-Glutamyl Carboxylase. Blood, 89(11), 4058–4062. https://doi.org/10.1182/blood.V89.11.4058

11.  Schuettrumpf, J., Herzog, R. W., Schlachterman, A., Kaufhold, A., Stafford, D. W., & Arruda, V. W. (2005). Factor IX variants improve gene therapy efficacy for hemophilia B. Blood,105(6), 2316–2323. https://doi.org/10.1182/blood-2004-08-2990