Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. This invention describes a strategy which simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, employing homologous recombination in bacteria rather than in eucaryotic cells. Following transfections of such plasmids into a mammalian packaging cell line, viral production can be conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system expedites the process of generating and testing recombinant adenoviruses.

https://www.pnas.org/content/pnas/95/5/2509.full.pdf

A simplified system for generating recombinant adenoviruses

The development over the past decade of methods for delivering genes to mammalian cells has stimulated great interest in the possibility of treating human disease by gene-based therapies. However, despite substantial progress, a number of key technical issues need to be resolved before gene therapy can be safely and effectively applied in the clinic. Future technological developments, particularly in the areas of gene delivery and cell transplantation, will be critical for the successful practice of gene therapy.

https://science.sciencemag.org/content/sci/260/5110/926.full.pdf

The Basic Science of Gene Therapy

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

Enough information has been gained from clinical trials to allow the conclusion that human gene transfer is feasible, can evoke biologic responses that are relevant to human disease, and can provide important insights into human biology. Adverse events have been uncommon and have been related to the gene delivery strategies, not to the genetic material being transferred. Human gene transfer still faces significant hurdles before it becomes an established therapeutic strategy. However, its accomplishments to date are impressive, and the logic of the potential usefulness of this clinical paradigm continues to be compelling.

Transfer of Genes to Humans: Early Lessons and Obstacles to Success

Polyamidoamine cascade polymers mediate efficient transfection of cells in culture

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium

The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin (alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.

https://science.sciencemag.org/content/sci/252/5004/431.full.pdf

Masashi Fukayama

Papers

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium

Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo

Respiratory tract gene transfer. Transplantation of genetically modified T-lymphocytes directly to the respiratory epithelial surface.