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How Gene Therapy Is Like a Weeble

By Anna Lau, PhD, Medical Writer

My Weebles collection. Don’t judge. WEEBLES is a registered trademark of Hasbro, Inc.

My Weebles collection. (Don’t judge.)

In 1963, at the dawn of the molecular biology age, Dr. Joshua Lederberg anticipated “the interchange of chromosomes and segments,” predicting that “the ultimate application of molecular biology would be the direct control of nucleotide sequences in human chromosomes, coupled with recognition, selection and integration of the desired genes…” His idea describes the basic principle of gene therapy. Less than three decades later, in 1990, the first report was published of successful retrovirus-mediated gene transfer in people. This raised hopes that genetic disorders would one day be cured by introducing functional genes into patients. But what ensued was a series of promising steps forward followed by steps back.

Jesse Gelsinger was a young man who suffered from ornithine transcarbamylase deficiency, an X-linked disorder that affects protein metabolism. In 1999, Jesse was enrolled in a pilot study to assess the safety of gene therapy in children with genetic diseases. He received a dose of adenoviral vector containing a working copy of the defective gene that caused his disease. Jesse developed a systemic inflammatory response that none of the first 17 patients in the pilot study had. He died several days later.

Like a Weeble, gene therapy wobbled, but it didn’t fall down. Researchers never gave up refining technologies.

The tragic death in 1999 of a young man from an acute innate immune response to an adenoviral vector spurred researchers to explore safer, less immunogenic vectors, like adeno-associated viral (AAV) and lentiviral vectors. The newer vectors are also preferable over retroviral vectors, which can integrate willy-nilly into the host genome, and cause insertional mutagenesis leading to cancer.

Schematic of the CRISPR/cas system. Cas9 (light blue blob) is a bacterial DNA endonuclease. Guide RNA (blue strand) contains a sequence that matches the target DNA sequence to be cut, which is marked by an adjacent recognition sequence called a PAM motif (orange strands). Cas9 generates site-specific double-stranded DNA breaks that need repair. If donor DNA (purple strands with black homology regions) is provided during repair, then homology-directed repair can occur, and new DNA can be introduced at the break site.

Non-viral approaches are also under investigation that would circumvent issues related to viral vectors, including a transposon-based system and an exciting new system called CRISPR/cas. CRISPR/cas serves as an adaptive immune system in bacteria, but has been exploited by researchers to make precise changes to DNA sequences in an array of model organisms (plants, yeast, nematodes, zebrafish, and mice) and in human cells. More importantly, researchers have used CRISPR/cas to successfully correct genetic defects in mouse models of human diseases, tyrosinemia and congenital cataracts.

Other endonuclease-based methods of site-directed mutagenesis, namely ZFN (zinc finger nucleases) and TALEN (transcription activator-like effector nuclease), have been developed. These are not discussed here, however, because the customization needed to target ZFNs or TALENs to specific sites in the genome is considerably more cumbersome, compared with CRISPR/cas. Because ZFN is an older technology, it has been used safely to disable the major coreceptor, CCR5, for HIV entry into host T cells in HIV-positive patients.

Researchers are also targeting more “gene therapy-amenable” disorders…

Some disorders are more amenable than others to the gene therapy approach, depending on the tissue type affected, the amount of therapeutic protein needed, and “invisibility” to the immune system, among other factors. For instance, the CFTR gene defect that causes cystic fibrosis must be corrected specifically in the lungs to affect the lung phenotype of the disease, whereas the factor IX (F9) gene defect that causes hemophilia B need not be corrected in a particular tissue as long as therapeutic levels of coagulation factor are present in plasma.

How much function does a corrective gene need to restore to be clinically meaningful? That can vary by disorder. For instance, expression of factor IX at >5% of normal levels represents a mild form of hemophilia B, so striving to reach this expression level is a reasonable target. (This study strove for >3% of normal levels.) But it’s not known exactly how much functional CFTR protein would be needed to rescue a patient with cystic fibrosis (although one group suggests at least 25%).

…and more “gene therapy-amenable” organs.

The eye is an organ particularly suitable for gene therapy because it is small, accessible, and sequestered from the immune system. This so-called immune privilege means that local immune and inflammatory responses are limited. There are about a dozen ongoing clinical studies of gene therapy for genetic eye disorders (some of which are described here).

Still, most gene therapy clinical studies are in cancer.

Cancer is the second leading cause of death in the US, so there is tremendous interest in applying knowledge of cancer molecular biology and genetics to finding its cure. As of June 2012, 1843 gene therapy clinical studies worldwide had been completed, were ongoing, or were approved to start. Of these, 1186 (64%) were studies in cancer. Gene therapy has been used with a wide variety of cancers in adults and in children, including gastrointestinal, gynecologic, lung, neurologic, skin, and urologic solid tumors and hematologic malignancies. The strategies used against cancer have included insertion of tumor suppressor genes, immunotherapy, oncolytic virotherapy, and gene-directed enzyme prodrug therapy.

Distribution of topics in gene therapy clinical studies, as of June 2012.

Investors are catching on to gene therapy. Will everyone else follow suit?

Since the beginning of 2013, biotechnology and pharmaceutical firms exploring gene therapy approaches have raised hundreds of millions of dollars in capital to fund their research. Now, not only must researchers overcome biological barriers to successful gene therapy (achieving sufficient gene transfer in target tissue, sustaining expression over time, limiting immune response to the gene delivery system, etc). Companies will also need to figure out how to scale up gene therapy approaches, price these approaches appropriately, and convince doctors, patients, and insurance carriers that gene therapy is a safe, efficacious, cost-effective approach to treatment.

In 2010, a new generation of Weebles was introduced, marking their comeback nearly 40 years after the originals. Dr. Lederberg’s prediction for gene therapy will take longer than that to be fulfilled—perhaps by 2023, six decades after his original prediction? Fingers crossed.

WEEBLES is a registered trademark of Hasbro, Inc.