Item Details

abstract

BACKGROUND: Children born with hemophilia A (HA) suffer tremendously due to frequent and spontaneous bleeding. The condition is caused by a mutation in the factor VIII (FVIII)-encoding gene. The standard of care for HA patients is replacement FVIII, infused 2-3 times per week, for their entire lives. Unfortunately, this therapeutic approach often leads to an anti-factor VIII response, subjecting patients to subsequent bleeding. A treatment approach that provides long-term, therapeutic FVIII, without eliciting an anti-FVIII response, remains outstanding.OBJECTIVE: To investigate the therapeutic adequacy of a prenatal transplant (IUTx) as an approach to HA treatment. This work was divided into four chapters. The first chapter reviewed clinical and experimental work detailing understandings of immune tolerance induction to therapeutic product in IUTx. The second chapter determined the ideal cellular platform for producing FVIII. The third chapter evaluated the effects of the cell platform (PLC-mcoET3) after prenatal injection in sheep fetuses and assessed IUTx recipients for over 36 months of life. The fourth chapter tested the effect of postnatal dosing of the same cell platform, during adulthood, in IUTx recipients and identified differential immune cell signaling pathways that respond to the dosed FVIII/ET3, when the product is cell-secreted versus direct injected as a protein. RESULTS: Results demonstrated that human placental cells (PLC), transduced to express a codon optimized, hybrid FVIII (mcoET3), produce clinically-meaningful FVIII levels in vitro, in absence of oncogenic activation or cytosolic stress. Then, after performing IUTx using the above-described cell platform (PLC-mcoET3), therapeutic FVIII levels in circulation of sheep (~3kg at birth, and > 80kg throughout adulthood) sufficient to convert severe HA to mild HA, were observed at all timepoints until at least 36 months of age, in the absence of anti-mcoET3/anti-PLC responses. Lastly, postnatal dosing of IUTx recipients with PLC-mcoET3 (20 IU FVIII / kg / 24 hrs.) did not significantly alter the sustained, elevated FVIII levels, and multiplex gene expression analysis revealed the upregulation of biomarkers associated with immune tolerance signaling, post-dosing. No significant anti-mcoET3 or anti-PLC responses ensued post-dosing in this group. In contrast, postnatal dosing with purified ET3 (20 IU FVIII / kg) led to a significant decrease in FVIII levels in one IUTx recipient. Moreover, postnatal dosing with ET3 protein triggered a high-titer, neutralizing anti-ET3 antibody and anti-ET3 effector T cell response in multiple animals. CONCLUSION: These results provide compelling evidence that the prenatal transplantation (IUTx) of PLC-mcoET3 represents a safe and promising treatment for HA, as this approach has the potential to achieve long-term phenotypic correction in the absence of anti-FVIII responses. Continued research in this area will provide a more complete understanding of the benefits and immune implications of prenatal exposure to therapeutic product via IUTx, thereby paving the way for the correction of a wide range of additional genetic diseases prior to birth.

subject

Clotting Factor VIII

Hemophilia A

Immune Tolerance

In Utero Transplantation

Placental Cell Therapy

Somatic Cell Gene Therapy

contributor

Rodriguez, Martin (author)

Almeida-Porada, Graça (committee chair)

Haas, Karen M (committee member)

Atala, Anthony (committee member)

Spencer, H. Trent (committee member)

Porada, Christopher (committee member)

date

2022-07-11T19:17:46Z (accessioned)

2022 (issued)

degree

Molecular Medicine and Translational Science (discipline)

2027-05-14 (liftdate)

embargo

2027-05-14 (terms)

identifier

http://hdl.handle.net/10339/101030 (uri)

language

en (iso)

publisher

Wake Forest University

title

PRENATAL CELL/GENE THERAPY FOR THE TREATMENT OF HEMOPHILIA A

type

Dissertation