Biology subjects

Freitag, P. C.

Publications and source records attributed to Freitag, P. C..

4 recordsLinked to original sources

bioRxiv2025

Genome-replicating HC-AdV, a novel high-capacity adenoviral vector class featuring enhanced in situ payload expression

High-capacity adenoviral (HC-AdV) vectors offer large transgene capacities and long-term expression of therapeutics, but require high doses due to limited transgene expression. In contrast, replication-competent AdV (RC-AdV) vectors enhance in situ transgene expression by genome replication and increased transcription from amplified genomes. Yet, RC-AdVs are constrained by minimal payload capacity, progeny formation, and toxic protein expression leading to rapid host cell death. To address these limitations, we developed a novel, genome-replicating HC-AdV vector. Therefore, we investigated AdV genome replication independently of progeny particle formation, and developed cell-based trans-replication assays, enabling us to probe the requirement for individual AdV proteins in AdV genome replication. We identified seven AdV proteins from the early transcriptional units which promote potent replication of HC-AdV genomes. We then created a genome-replicating HC-AdV vector by encoding an engineered minimal replication system that functionally reconstitutes AdV genome replication. Host cell transduction with our genome-replicating HC-AdV promoted cis-replication of the delivered HC-AdV genome and up to 20-fold increased reporter fluorescence. Our novel vector retained a large transgene capacity (22 kb) and, unlike RC-AdVs, did not induce a cytopathic effect nor host cell killing. Together, these data describe a novel delivery platform potentially allowing more efficacious vaccination and vector-mediated therapies.

biochemistry↗

bioRxiv2024

Dendritic cell targeting in lymph nodes with engineered modular adapters improves HAdV5 and HC-HAdV5 tumor vaccination by co-secretion of IL-2v and IL-21

Adenoviral vectors demonstrate encouraging clinical outcomes for B- and T-cell vaccines. With such approaches, multiple payloads can be delivered, beyond the antigen itself. Nevertheless, the human adenoviral vector serotype C5 (HAdV5) exhibits limited transduction efficiency to dendritic cells (DC), therefore necessitating very high viral loads. Targeting antigen-presenting cells (APC) has remained challenging. To solve this problem, we developed a versatile platform that employs modular retargeting adapters to enhance transduction of specific cell types, including challenging host cells. By rational design, we constructed a dual-adapter for DC-SIGN and CD11c and demonstrate successful targeting of HAdV5 to human and murine DCs. Our in vivo characterization highlights improved and specific transduction of DCs in draining lymph nodes. Moreover, a tumor vaccination study showcases the advantageous co-expression of T cell stimulatory cytokines (IL-2v or IL-21) locally in lymph nodes alongside a potent tumor antigen. Lymph node-directed gene therapy at significantly reduced vector loads circumvents potential systemic toxicity of stimulating payloads. Our proposed low-dosage DC-targeted vaccine offers an effective solution for patients and also minimizes potential adenovirus-related side-effects. The robust immunogenicity of HC-HAdV5, with its large coding capacity (37 kbp DNA), opens up exciting possibilities for future therapeutic combination strategies.

biochemistry↗

bioRxiv2023

Targeting HIV-1 with CRISPR/Cas9 delivered by retargeted adenoviruses effectively suppresses viral replication

Integrated, intact, latent HIV-1 viruses in infected cells are the main obstacle to curing HIV-1 infections. Targeted inactivation of HIV-1 proviruses with CRISPR/Cas9 is a promising strategy to eradicate HIV-1. In addition, CRISPR/Cas9 is able to target replicating HIV-1 and could be used as a therapy during productive infection. Here, we combine the CRISPR/Cas9 system with a novel adenovirus (Ad) targeted delivery technology to test it as a therapeutic approach to inhibit HIV-1. First, we selected six HIV-1-specific gRNAs targeting the HIV-1 LTRs and the gag gene and tested their efficacy in inhibiting HIV-1 virion production in an HEK 293T cell co-transfection screen. The gRNA-TAR showed the most robust and potent inhibition of HIV-1 by >99% alone or in combination with the gRNA-p24, which induced a [~]1 kb deletion between both gRNA target sites in HIV-1 DNA. Delivery of this dual gRNA-TAR/p24 CRISPR/Cas9 system with CD3-CD28-IL2-retargeted Ads was highly effective, transducing 62.3{+/-}23.3% of cells and suppressing HIV-1 replication by 88.0{+/-}4.5% in primary CD4+ T cells from three independent donors. Our dual gRNA-TAR/p24-CRISPR/Cas9-Ad strategy represents a novel therapeutic approach to effectively inhibit HIV-1 in a highly HIV-1 and T cell-specific manner.

microbiology↗

bioRxiv2022

FAP-retargeted Ad5 enables in vivo gene delivery to stromal cells in the tumor microenvironment

Fibroblast activation protein (FAP) is a cell surface serine protease that is highly expressed on reactive stromal fibroblasts, such as cancer-associated fibroblasts (CAFs), and generally absent in healthy adult tissues. FAP expression in the tumor stroma has been detected in more than 90% of all carcinomas, rendering CAFs excellent target cells for a tumor site-specific adenoviral delivery of cancer therapeutics. Here, we present a tropism-modified human adenovirus 5 (Ad5) vector that targets FAP through trivalent, designed ankyrin repeat protein (DARPin)-based retargeting adapters. We describe the development and validation of these adapters via cell-based screening assays and demonstrate adapter-mediated Ad5 retargeting to FAP+ fibroblasts in vitro and in vivo. We further show efficient in vivo delivery and in-situ production of a therapeutic payload by CAFs in the tumor microenvironment (TME), resulting in attenuated tumor growth. We thus propose using our FAP-Ad5 vector to convert CAFs into a biofactory, secreting encoded cancer therapeutics into the TME to enable a safe and effective cancer treatment.

biochemistry↗