Leveraging gene therapy to achieve long-term continuous or controllable expression of biotherapeutics

TIMOTHY P. CRIPE HTTPS://ORCID.ORG/0000-0002-8595-3577 BRIAN HUTZEN[...]PIN-YI WANG  Authors Info & Affiliations
13 Jul 2022
Vol 8, Issue 28
DOI: 10.1126/sciadv.abm1890


T cells redirected to cancer cells either via a chimeric antigen receptor (CAR-T) or a bispecific molecule have been breakthrough technologies; however, CAR-T cells require individualized manufacturing and bispecifics generally require continuous infusions. We created an off-the-shelf, single-dose solution for achieving prolonged systemic serum levels of protein immunotherapeutics via adeno-associated virus (AAV) gene transfer. We demonstrate proof of principle in a CD19+lymphoma xenograft model using a single intravenous dose of AAV expressing a secreted version of blinatumomab, which could serve as a universal alternative for CD19 CAR-T cell therapy. In addition, we created an inducible version using an exon skipping strategy and achieved repeated, on-demand expression up to at least 36 weeks after AAV injection. Our system could be considered for short-term and/or repeated expression of other transgenes of interest for noncancer applications.


Most medications are administered on an intermittent schedule, resulting in pharmacokinetic peaks and troughs. Great efforts are expended to find dosages and schedules that minimize time spent outside of the therapeutic window, resulting in more frequent doses for drugs with shorter half-lives, inevitably with troughs below efficacious levels. These troughs are a particular challenge for cancer, as they enable periods of cancer regrowth and development of drug resistance. Furthermore, drugs with extremely short half-lives require continuous infusions, which are cumbersome as they require frequent hospital or home care visits for bag or syringe changes, pose vascular access challenges, and create risk for infections. Diseases such as cancer, cardiovascular disease, and chronic pain require long-term therapy, and others such as rheumatologic diseases require intermittent therapy over long periods of time.
Recent advances in gene therapy for correction of monogenic disorders have led to successful replacement of missing or defective genes, yielding constant expression of a transgene for months to years. We sought to determine whether gene therapy could also potentially solve the problems of peaks and troughs for protein-based medicines by enabling sustained and consistent levels of therapeutics in the blood and tissue after a single dose.
As a proof of principle of gene-based therapeutics for cancer, we designed recombinant adeno-associated virus (rAAV) vectors to express a bispecific T cell engager. These proteins are typically composed of two different antibody single-chain variable fragments (scFv) connected by a linker [a bispecific tandem diabody (1)] and transiently join T cells to target cells, promoting T cell activation and target cell apoptosis. The most advanced of these agents is blinatumomab, composed of an anti-CD19 scFv linked to an anti-CD3 scFv, which is currently Food and Drug Administration (FDA)–approved for CD19+ B cell acute lymphoblastic leukemia (ALL) in children and adults who have either relapsed or refractory disease or who have minimal residual disease after first or second remission. The short half-life of blinatumomab [~2.1 hours; (2)] necessitates its continuous intravenous infusion over a period of 2 to 6 months (with intermittent breaks). As a consequence, the full utility of this therapeutic to subject cancer cells to consistent, long-term pressure has not been realized.
We created an rAAV with a human codon-optimized transgene encoding a signal peptide followed by the amino acid sequence of blinatumomab, which we term CD19 TransJoin, named for expression of a transgene that joins two cells together. We refer to the secreted protein product produced by the virus as an anti-CD19–anti-CD3 (αCD19-αCD3) “dimert.” In addition to only requiring a single dose, our system also enables prolonged pressure on the target tumor cell, resulting in therapeutic expression for more than 1 year, a feature thought to be important for long-term cures elicited by chimeric antigen receptor (CAR) T cell approaches. Unlike CAR-T cells, however, TransJoin is an off-the-shelf solution, circumventing cumbersome manufacturing challenges inherent to CAR-T therapy.
To address clinical scenarios that require only short-term expression or recurring, intermittent expression of a therapeutic, we also developed a companion AAV construct termed TransSkip, in which we inserted two introns flanking a defective exon into the transgene coding sequence. Thus, constitutive exon inclusion in untreated samples renders the transgene nonfunctional. Upon administration of a morpholino complementary to the 3′ exon splice site, the exon is skipped, resulting in alternate splicing that restores full-length transgene expression. TransSkip, therefore, represents an inducible gene therapy modality.