(452e) Transcriptome Analysis of the Host Cell Response to Non-Viral Gene Therapy | AIChE

(452e) Transcriptome Analysis of the Host Cell Response to Non-Viral Gene Therapy


Tucker, M. - Presenter, Villanova University
Elmer, J., Villanova University
Transcriptome Analysis of the Host Cell Response to Non-Viral Gene Therapy

Although several highly efficient viral and non-viral vehicles have been developed to deliver genes to cells, transgene expression levels tend to be too low to provide the desired therapeutic response in eukaryotic cells. It has been previously suggested that transgene expression may be actively inhibited by the innate immune response and other mechanisms within the host cell. Indeed, many vehicles deliver genes to the cytoplasm, where DNA is recognized as a sign of viral/bacterial infection (since all the DNA in a healthy cell is sequestered in the nucleus). The cytoplasmic DNA is bound by one of several different DNA sensor proteins, which activate signaling cascades that trigger the expression of many different defensive genes (e.g., cytokines, transcription factors, et al.) that limit transgene expression by inhibiting transcription and translation or inducing apoptosis or pyroptosis. Consequently, the host cell response to gene delivery is a crucial factor that must be considered when designing gene therapy treatments.

While previous studies have used ELISA, rtPCR, and other methods to identify the upregulation of specific genes (e.g., IL-6 & CXCL10) in response to gene delivery, next-generation sequencing was used in this work to interrogate the entire transcriptome of the host cell and identify all of the host cell genes that are up- or down-regulated in response to cytoplasmic delivery of plasmid DNA. Specifically, RNA-Seq was used to quantify changes in the transcription of both mRNA and small non-coding RNAs (e.g., miRNA & snoRNA) following delivery of plasmids with non-viral vehicles (Lipofectamine and polyethyleneimine) to multiple cell types (prostate cancer PC-3, breast cancer MCF7, and Jurkat T-cell leukemia cells).

As expected, several cytokines (IL-6, IFN b, CXCL10, et al.) and transcription factors (e.g, STAT1, IRF7, SP8, ELF4, ATF3) were upregulated by at least 10-fold. Some previously unidentified genes were also upregulated, including IFN λ, CXCL11, AIM2, GTPases, PARPs, histone isoforms, and a set of ubiquitinylation enzymes. Several additional genes with unknown function were also upregulated by ≥10-fold, including hemoglobin delta, keratins, CSAG 1/2/3, and several interferon-inducible proteins. Gene Set Enrichment Analysis (GSEA) was employed to identify specific pathways involving the upregulated genes and confirmed the activation of multiple redundant innate immune response pathways. Subsequent ELISAs also confirmed increases in the expression of specific cytokines (e.g. IL-6) after transfection. Since we have previously shown that the β-catenin/TCF4 inhibitor iCRT14 can increase transgene expression by altering cytokine expression profiles, we also investigated the effects of iCRT14 on the transcriptome to identify key genes involved in transgene repression that can be inhibited by iCRT14. In conclusion, our results help to define the entire host cell response to non-viral gene therapy and identify several key inflammatory and inhibitory genes/pathways that should be considered (and perhaps inhibited) when designing gene therapy treatments.