Biodegradable nanoparticles for drug and gene delivery to cells and tissue

An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes.

https://www.pnas.org/content/pnas/91/10/4407.full.pdf

Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy

Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis

Endocrine Regulation of the Fasting Response by PPARα-Mediated Induction of Fibroblast Growth Factor 21

Transcription termination and 3' processing: the end is in site!

Accumulation of vascular smooth muscle cells as a consequence of arterial injury is a major feature of vascular proliferative disorders. Molecular approaches to the inhibition of smooth muscle cell proliferation in these settings could potentially limit intimal expansion. This problem was approached by introducing adenoviral vectors encoding the herpesvirus thymidine kinase (tk) into porcine arteries that had been injured by a balloon on a catheter. These smooth muscle cells were shown to be infectable with adenoviral vectors, and introduction of the tk gene rendered them sensitive to the nucleoside analog ganciclovir. When this vector was introduced into porcine arteries immediately after a balloon injury, intimal hyperplasia decreased after a course of ganciclovir treatment. No major local or systemic toxicities were observed. These data suggest that transient expression of an enzyme that catalyzes the formation of a cytotoxic drug locally may limit smooth muscle cell proliferation in response to balloon injury.

https://science.sciencemag.org/content/sci/265/5173/781.full.pdf

Gene Therapy for Vascular Smooth Muscle Cell Proliferation After Arterial Injury

We have employed an antiserum specific to the 70-kilodalton human heat shock protein and a cDNA clone specific to the mRNA for this protein to analyze the expression of the gene under noninducing conditions. Expression of the heat shock gene can be detected in the absence of heat induction, and this uninduced level of expression depends greatly on the particular cell type. For instance, the basal expression of the heat shock gene is at least 50 times higher in HeLa cells than in WI38 cells at both the mRNA and protein levels. We have previously shown that the inducer of transcription of the early adenovirus genes, the E1A gene product, also induces the heat shock gene, suggesting that these genes may be subject to the same regulation. We have, therefore, investigated the control of the adenovirus genes in relation to the cellular control of the heat shock gene. We find that human cells that allow a high level of uninduced expression of the heat shock gene (i.e., HeLa cells) also allow expression of the early adenovirus genes in the absence of the E1A inducer. The same is also true for the mouse F9 teratocarcinoma cell line. F9 stem cells, which constitutively express the heat shock protein, allow early adenovirus gene expression in the absence of E1A; upon differentiation induced by retinoic acid and cyclic AMP, the cells become restrictive and early viral gene expression requires the E1A gene product. Coordinately, upon differentiation there is also a loss of heat shock protein expression.

Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity.

One of the major limitations to current gene therapy is the low-level and transient vector gene expression due to poorly defined mechanisms, possibly including promoter attenuation or extinction. Because the application of gene therapy vectors in vivo induces cytokine production through specific or nonspecific immune responses, we hypothesized that cytokine-mediated signals may alter vector gene expression. Our data indicate that the cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) inhibit transgene expression from certain widely used viral promoters/enhancers (cytomegalovirus, Rous sarcoma virus, simian virus 40, Moloney murine leukemia virus long terminal repeat) delivered by adenoviral, retroviral or plasmid vectors in vitro. A constitutive cellular promoter (β-actin) is less sensitive to these cytokine effects. Inhibition is at the mRNA level and cytokines do not cause vector DNA degradation, inhibit total cellular protein synthesis, or kill infected/transfected cells. Admin...

Promoter Attenuation in Gene Therapy: Interferon-γ and Tumor Necrosis Factor-α Inhibit Transgene Expression

The Role of Polyomaviruses in Human Disease

In the past decade, adenovirus vectors have generated tremendous interest, especially in gene therapy applications. In the so-called 'first generation' adenovirus vectors, the transgenes are inserted in place of the E1 region, or less often the E3 region. Although second-generation and helper-dependent adenovirus vectors will probably prevail in the future in applications that require long-term gene expression, first generation adenovirus vectors will remain very useful in other settings, such as cancer and vaccination, or simply to transfect cell lines that are refractory to other transfection methods. Until a few years ago, the construction of first generation adenovirus vectors was a labor-intensive and time-consuming process. More than 20 methods have appeared that facilitate their construction and are reviewed below.

Michael J. Imperiale

Papers

Gene Therapy for Vascular Smooth Muscle Cell Proliferation After Arterial Injury

Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity.

Promoter Attenuation in Gene Therapy: Interferon-γ and Tumor Necrosis Factor-α Inhibit Transgene Expression

The Role of Polyomaviruses in Human Disease

Production of first generation adenovirus vectors: a review