Retroviral RLI immunomodulatory gene therapy for glioblastoma

PROJECT SUMMARY/ABSTRACT
Antitumor immune responses require a functional repertoire of innate and adaptive immune cells. Glioblastoma
(GBM), however, harbors a profoundly immunosuppressed microenvironment, particularly its T cell ignorance
caused by bone marrow sequestration; T cell exhaustion caused by immune checkpoint molecules on the
surface of T cells that suppress T cell function; and impaired memory T-cell responses. Unfortunately, efforts to
target the immunosuppressed GBM microenvironment with systemic immunotherapies have not produced
meaningful impact in clinical trials. Localized viral treatments have also been investigated for GBM and, while
these viruses elicit an anti-tumoral immune response, these treatments have also failed to impact survival in
clinical trials. To address these limitations, we have investigated intratumoral delivery of a replicating retrovirus
expressing RLI, which encodes an interleukin-15 fusion protein that enhances CD8+ and CD4+ naïve and
memory T-cell proliferation, as a therapeutic strategy free of the toxicities of systemic treatments targeting the
tumor microenvironment. We demonstrated that replicating retroviral delivery of RLI prolonged survival of
immunocompetent mice with intracranial gliomas using multiple different models. Here, we will build upon our
data by investigating our central hypothesis that intratumoral RLI immunomodulatory gene therapy can be
potentiated by adding other immunomodulatory strategies, incorporating immunogenic cell death, or targeting
resistance mechanisms. We will investigate our hypothesis through four specific aims: (1) Potentiate RLI
immunomodulatory gene therapy by enhancing T-cell mobilization, co-stimulation, and memory; (2) Determine
if targeting checkpoint pathways potentiates retroviral RLI immunomodulatory gene therapy; (3) Enhance RLI
immunomodulatory gene therapy by incorporating immunogenic cell death; and (4) Identify and target
glioblastoma-expressed proteins that counteract retroviral RLI immunomodulatory gene therapy. Our pursuit of
these aims will utilize novel technologies developed by our lab such as our binary retroviral system to deliver a
large payload of immunomodulatory genes and our retroviral compact Cas13d RNA-targeting CRISPR to target
resistance mechanisms. We will combine these innovative approaches with cutting-edge technologies such as
CyTOF to characterize the effects of RLI-based retroviral therapies on the full cohort of innate and adaptive
immune responses; customized CRISPRi libraries; paired immunodeficient and immunocompetent mice strains
to isolate immunologic resistance mechanisms; and single cell sequencing to profile T-cell subsets altered by
these therapies. These studies will develop our novel localized RLI retroviral immunotherapy in a manner that
addresses the spectrum of mechanisms creating local and systemic immunodeficiency in GBM by accounting
for T-cell ignorance and exhaustion, and identifying and targeting tumor cell and immune cell-driven resistance
mechanisms before they evolve. In doing so, we will validate our hypothesis regarding the impact of RLI-based
immunomodulatory gene therapy on GBM, a novel strategy with significant translational potential.

Grant Number: 5R01NS123808-04
NIH Institute/Center: NIH
Principal Investigator: Manish Aghi