Gene Theraphy Emerges From Disgrace to Be the Next Big Thing Again

Biotechnol Healthc. 2005 Jun; 2(three): 52-54, 56-sixty.

The Time to come of Gene Therapy

Abstruse

Eventually, gene therapy will become a staple of 21st century medicine. But some experts say society will be better served if medical researchers continue more slowly and prudently.

In its electric current manifestation, factor therapy is an elegant concept crudely executed. That's non an indictment — that'southward just the way it is for an extraordinarily complicated engineering still in its infancy. After all, it has been simply v years since the concept of gene therapy was convincingly demonstrated to provide, if non a cure, then at least a long-term therapeutic upshot for X-linked astringent combined immunodeficiency (X-SCID) disease.

(Hither, gene therapy is divers as the introduction of genetic material via techniques of molecular biology into somatic cells [in contrast to germ cells] to treat or foreclose disease.)

RELATIVELY Brief HISTORY

Many people still are nether the impression that gene therapy's proof-of-concept was demonstrated every bit early equally 1990. For example, on Jan. 26 of this year, the Los Angeles Times wrote that Due west. French Anderson, MD, was "dubbed 'the father of cistron therapy' after a team he led in 1990 cured a hereditary disease of the immune system in a 4-year-quondam girl." That'due south not quite the way it happened.

Anderson did indeed proceeds renown for heading the team that in September 1990 carried out the first gene therapy clinical trial approved for use in a human. The goal of this phase one report was to ascertain the prophylactic issues involved. The four-yr-quondam girl had a genetic disease called adenosine deaminase (ADA) deficiency, which is caused past a defective factor for the enzyme ADA, resulting in SCID. Via a modified retrovirus, normal ADA genes were transferred to T lymphocytes that had been removed from the daughter's body and grown in culture. The white cells so were returned to the patient. In January 1991, a ix-yr-old girl underwent the same procedure.

"The concept of fixing a broken gene is at present entrenched in medicine," says Theodore Friedmann, Md, director of the University of California–San Diego Plan in Gene Therapy. "[It'south] a real field, with many ups and downs behind it and surely more than alee. Its engineering science is evolving, slowly but certainly positively."

Photograph By ROBERT BURROUGHS

Today both patients are live and doing well, but conventional therapy (pegylated bovine ADA, or PEG-ADA) given before, during, and after their factor therapy confounded the results and makes any claim of "cure" based on the gene therapy problematic. 1 patient has an ADA level that is 25 percent of normal with the therapeutic gene nowadays in xv percentage of her peripheral claret mononuclear cells. The other has an ADA level that is less than 5 percent of normal, and the presence of the therapeutic cistron in the peripheral blood cells is negligible.

Anderson went on to interact in 12 of the first 20 cistron therapy trials canonical in the United States. In an article he wrote about the prospects for gene therapy, he stated, "Indeed, within 20 years, I expect that cistron therapy will exist used regularly to amend —and fifty-fifty cure — many ailments." More recently, Time celebrated the 50th ceremony of Watson and Crick's 1953 discovery of the structure of Dna with a special outcome about genetics, over again featuring Anderson. E'er the optimist, Anderson looked 50 years ahead and told Time, "By 2053, in that location volition exist a factor-based treatment for substantially every disease. Cancer, heart disease, and other modern-twenty-four hour period scourges will be vastly reduced."

Halfway through Anderson'southward twenty-yr window (just barely into his l-yr projection), yous tin count the number of clearly effective gene transfer therapies in nonexperimental clinical use with your nose. That's correct — there'south one. And you take to go halfway around the world to go it.

In October 2003, in China, Gendicine became the world's starting time cistron therapy approved for commercial product. According to newspaper accounts, patients from around the world accept been traveling there to receive this handling for head and neck squamous cell carcinoma (HNSCC). The question is how long volition Gendicine remain the world's only licensed factor therapy?

Probably, not very long — the Chinese approach is conceptually identical to one that was developed earlier in the The states merely has been moving more slowly toward licensing — a pace that means gene therapy will not come into regular utilise anytime presently.

On the other manus, the number and variety of clinical trials of gene therapy are such that Anderson's fifty-year project could pan out. As of December 2004, 667 homo cistron transfer clinical protocols had been submitted for review by the National Plant of Health'due south Recombinant Dna Informational Commission (RAC)¹ and the U.S. Food and Drug Administration (through its Center for Biologics Evaluation and Research). Of these, 617 are for therapeutic purposes (see table on page 56) — as opposed to marker and nontherapeutic purposes — but only a handful have advanced to phase 3. Most are phase 1 trials whose purpose is to demonstrate safety. Of the 59 protocols submitted in 2004, the majority originated in academia, simply 37 percent had a corporate sponsor (sometimes working in collaboration with a nonprofit grouping).

MULTITUDE OF TARGETS

With all the hoopla surrounding the Human Genome Project, it's understandable that people would entertain high hopes for the advancement of gene therapy. The human genome is now known to contain some 25,000 genes, including about 22,000 poly peptide-encoding genes that express about 100,000 proteins. But unresolved questions grow regarding what these genes and proteins really practise and how, when, where, and in what sequence. As answers emerge, gene therapy could evolve in ways that volition provide numerous benefits to patients and without deleterious side effects.

Talking nigh gene therapy equally though it were a single entity, though, isn't very helpful. As explained by David A. Sanders, PhD, associate professor of biological sciences at Purdue Academy, gene therapy falls into three groups:

• Replacing a lacking or mal-adaptive factor that's responsible for some monogenic affliction (e.g., cystic fibrosis or sickle cell anemia)

• Altering or killing an abnormal cell (e.g., infected past HIV or cancerous)

• Inducing production of a therapeutic poly peptide (e.yard., treating hepatitis C past promoting secretion of interferon by other cells)

Initially, gene therapy focused on the first group, only most current inquiry focuses on the other 2. Whatsoever the application, numerous hurdles stand up in the way of developing a successful cistron therapy, such as:

• Identifying an advisable target for gene therapy

• Getting a therapeutic transgene into the right cells (and only those cells) in the correct amount

• Delivering the transgene with a vector that doesn't trigger an immune response or, in the case of certain viral vectors, revert to a pathogenic form

• Providing the appropriate regulatory elements for turning the gene on and off at the correct time

• Keeping the transgene in the target cell long enough for it to do its chore

• Keeping the transgene from causing damage elsewhere (e.1000., spurring evolution of neoplasms or autoimmune illness, which could happen if the transgene expresses a poly peptide new to the patient's body

Aside from the one,500 or so diseases known to be acquired past a single defective factor, most involve multiple genes, so potential targets for gene therapy abound. Possible therapies aren't restricted to the naturally occurring genes and gene products in the man genome. Fusion genes and nonhuman genes also are being investigated. An example of a fusion factor is an investigational amanuensis being developed by Targeted Genetics, based in Seattle, Wash. This agent fuses the cistron for the Fc fragment of human immunoglobulin G with another factor for the soluble p75 receptor for tumor necrosis cistron, producing the same molecule provided by etanercept (Enbrel). A phase 1 trial comprising patients who have rheumatoid arthritis was initiated in March 2004.

Sky-HIGH EXPECTATIONS

In the view of Theodore Friedmann, Dr., who has been deeply involved in the study of gene therapy for three decades — substantially its entire modern history — hyperbole surrounding early on claims had the effect of unrealistically heightening expectations that gene therapy would emerge quickly every bit a component of health care. Friedmann is a professor of pediatrics at the Academy of California–San Diego, and managing director of the UCSD Program in Gene Therapy. A medical ethicist and geneticist, he as well has served on the RAC and was its chairman until last year.

That the initial human cistron transfer protocol was hailed a success, Friedmann says, is a "perfect example of the confluence of exaggerated expectations and wishful thinking. Everyone wanted it to work." Just, he adds, it was unfair to patients and the public that the heightened expectations generated past some of the scientists and their institutions, the media, and others served to raise simulated hope in many patients with many kinds of disease. "Hope is necessary, but knowingly making undeliverable promises and raising false hope is roughshod," Friedmann says. "The mirage of a cure contributed to crashing disappointment later."

With their early expectations dashed, people concluded that gene therapy might exist another biotech bosom. Friedmann thinks the perception of failure was also fueled by widely publicized setbacks being portrayed as "disasters" — which he says were regarded as disasters but because expectations were then loftier to commencement with.

Friedmann sees gene therapy today at a point comparable to the early on days of organ transplantation, when successes were deficient and failures frequent. Even the kickoff clear success of gene therapy, he notes, has been muted by the emergence of iii cases of leukemia (including one death) amidst the xviii children who were treated. The result of these cases is that gene transfer therapy is at present reserved for patients who accept had unsuccessful attempts at os marrow transplantation or for whom this approach is not viable.

"These children received very effective treatment, but at a very loftier cost," Friedmann says, "and if additional cases of leukemia develop, we'll have a greater trouble." But he adds, that doesn't mean that we should non button ahead with the field.

Human factor transfer protocols, Usa and worldwide, 1988–2005

	U.S. total	%	World total*	%
Cancer	436	65	675	66
Immunotherapy/in vivo transduction	159
Immunotherapy/in vitro transduction	129
Pro-drug/HSV-TK and ganciclovir	45
Tumor suppressor gene	38
Vector-directed cell lysis	28
Other therapeutic approaches	37
Monogenic Diseases	60	9	93	nine
Cystic fibrosis	23
Astringent combined immunodeficiency (SCID)	6
Hemophilia	5
Fanconi anemia	iv
Other monogenic diseases	22
Cardiovascular Disease	55	8	85	eight
Peripheral artery affliction	29
Coronary avenue disease	21
Other cardiovascular disease	5
Infectious Disease	42	half dozen	68	7
Man immunodeficiency virus	39
Other viral diseases	3
Central Nervous System Diseases	v	<1	5	<ane
(Alzheimer's disease, Parkinson'south disease, epilepsy)
Other Diseases & Disorders	19	3	26	three
(arthritis, autoimmune disease, bone fracture, cubital tunnel syndrome, erectile dysfunction, center disorders, intractable pain, peripheral neuropathy, ulcer)
Marking and Nontherapeutic Uses	50	7	68	7
Full gene transfer protocols	667		1,020

WHERE THE PATIENTS ARE

At get-go it was thought that gene therapy would focus on monogenic diseases — hereditary diseases such as SCID, hemophilia, or cystic fibrosis — that stem from a single defective gene. The thinking was that such diseases could exist ameliorated, if not cured, by providing the patient with a properly operation cistron. Thus far, the initial expectations have not been fulfilled. Moreover, as gene therapy has evolved, it has drifted abroad from monogenic diseases and toward diseases like cancer.

This makes sense given that cancer is where the patients are and probably volition be. Which is besides where the coin will exist. Co-ordinate to the American Cancer Gild, cancer has become the leading killer of Americans nether the age of 85, surpassing cardiovascular disease. For demographic reasons, it is logical that CVD also would attract the attention of gene therapy researchers — and information technology has, forth with incurable conditions such as Alzheimer'south illness and Parkinson's disease.

"Whatever the problems with sure drugs might be, they begin and terminate in a single generation," says David A. Sanders, PhD, acquaintance professor of biological sciences at Purdue University. In contrast, he points out, gene therapy has the potential to affect a patient for a lifetime, which mandates lifelong observation.

PHOTOGRAPH By SHAWN SPENCE

Alameda, Calif.-based Avigen began a stage 1/2 clinical trial of AV201 for the treatment of severe Parkinson's at the end of 2004. This agent uses an adeno-associated virus (AAV) vector to deliver the cistron for an enzyme, acetoacetate decarboxylase (AADC), directly into the striatum. The striatum is the section of the encephalon where move is controlled via dopamine. As Parkinson'southward progresses, dopamine levels decrease. Patients are treated with levodopa, which is converted to dopamine by AADC. Eventually, the effectiveness of levodopa diminishes, presumably considering the concentration of AADC declines. The idea behind AV201 is that with the enzyme restored, patients once again will respond to levodopa.

To have another case, some gene therapy research is exploring the function of angiogens — molecular mediators that promote the germination of blood vessels during normal cardiac and vascular development. 1 such angiogen is vascular endothelial growth factor (VEGF). The VEGF gene consists of eight exons that can exist spliced in different ways (omitting one or more exons), leading to the synthesis of amino acid sequences of varying lengths (specifically, 121, 165, 189, and 206 amino acids). Using an adenovirus vector, attempts have been made to transfer cDNA for VEGF121 into skeletal muscle as a treatment for peripheral arterial disease and into myocardial tissue as a treatment for severe coronary avenue affliction.

It was once thought that the receptors for the VEGF group of proteins were restricted to endothelial cells, but they since have been establish in cells of nonendothelial origin, including tumor cells. That finding points to a feature that cistron therapy shares with pharmacotherapy: the molecular targets of therapy often are not restricted to cells in the tissue of interest. That puts a premium on developing vectors that deliver transgenes to specific cells and only those cells.

IMPROVING VIRAL VECTORS

Among the numerous vehicles for carrying therapeutic genes to target cells, vectors adjusted from viruses stand out because of the ease with which viruses enter cells so spill out their contents — the viral genes that induce the host to generate the components of new virions. When a therapeutic gene is inserted in place of most of the viral genome, the virion retains its ability to penetrate the target cells while delivering a presumably beneficial payload. The families that have been near oft used as vectors are the adenoviruses and retroviruses, but AAV and lentivirus are among other viral vectors increasingly employed in gene therapy experiments. Lentiviruses actually are a genus in the retrovirus family unit, but they differ from other retroviruses in existence able to integrate their genome into the chromosomes of nondividing cells (e.g., brain, peripheral nerves). Other retroviruses can transduce only dividing cells.

Co-ordinate to Sanders, ane of Friedmann'due south major contributions, amid many, to the development of cistron therapy was showing that a recombinant pseudotyped virus could be created to serve equally a vector for delivering genetic cloth to cells. The genome of a pseudotyped virus lacks the coding for one or more of its structural proteins, which confers a safety benefit and other advantages.

In gimmicky factor therapy experiments, vesicular stomatitis virus G protein (VSV-Grand) pseudo-typed retroviruses and lentiviruses are commonly used but accept several shortcomings, such as being toxic to cells producing virus in civilisation and targeting primary cells in civilisation or in vivo. In his ain work, Sanders has been looking at other viruses that could be used to create pseudotyped viruses, and he says that he has institute alphaviruses to be promising. Among the species in this insect-transmitted genus are the Ross River virus, Eastern equine encephalitis virus, Semliki Wood virus, and Venezuelan equine encephalitis virus.

The field of factor therapy is driven largely past medical professionals instead of scientists. Because patients are their principal business organisation, doctors might be predisposed to trying gene therapy experiments on severely ill patients — even if the scientific discipline is still a bit ragged.

A protein biochemist by training, Sanders did postdoctoral work with Harvard's Richard Mulligan, PhD, who joins Friedman equally some other pioneer of factor therapy. Sanders is particularly interested in the proteins institute on the exterior of viruses, because the way these proteins match upwards with other proteins embedded in the cell membrane determines whether a virus tin enter a given prison cell. Sanders has designed a new class of pseudo-typed viruses, constructing their shell from a variety of alphaviruses and their core from retroviruses and lentiviruses. Injected into the tail vein of a mouse, these pseudotyped viruses are delivered in quantity to the liver. And in the central nervous system, these vectors become specifically to glial cells but non to neurons, in contrast with VSV-Thou vectors, which enter neurons but not glial cells. Sanders says this property has important implications for the treatment of brain tumors, about of which are of glial origin.

In many cases, delivering a gene to the right cells is not sufficient. The gene also must be brought to the correct portion of the jail cell. That's considering many cells are polarized, having one portion of their plasma membrane exposed to the outside world (the upmost membrane) and another exposed to the blood stream (the basolateral membrane). The proteins embedded in the apical membrane differ from those in the basolateral membrane, and a tight junction prevents proteins from passing from i domain to the other. Because unlike proteins offer a foothold for different viruses, it'south of import to select a viral vector that's specific for the apical or basolateral membrane of polarized target cells, such as airway epithelial cells in the lung. Influenza viruses enter and leave these cells through the apical membrane, which is exposed to the exterior. But a retrovirus similar HIV or murine leukemia would take to enter through the basolateral membrane, which is exposed to the bloodstream in which these viruses find their major target (claret cells).

Sanders explains that a applied consequence of failure to capeesh the difference between apical and basolateral membranes is this: More than a decade agone, the cistron responsible for cystic fibrosis was discovered. Great excitement ensued, and the success of gene therapy based on this discovery was eagerly anticipated. But success has been elusive, partly owing to difficulty in getting the therapeutic factor into the target cells. Sanders says that's considering researchers used retroviral vectors that approached the epithelial cells from the wrong side (the basolateral side). He says a more than promising approach may be to base a vector on a modified Ebola virus beat, which specifically targets airways epithelial cells and tin can enter through the apical membrane if it'due south aerosolized. This technique appears to better gene delivery by ii orders of magnitude compared to retroviral vectors, Sanders says.

Incorrect DRIVER?

Setting aside the specifics of cystic fibrosis, the greater question focuses on why researchers would use the wrong vector in the outset identify. In Sanders' view, it's because the field of gene therapy is driven largely by medical professionals instead of scientists. The md'due south paramount concern is to help a patient, and if the patient is severely ill, the doctor may disregard loose ends or ambiguity in the scientific discipline supporting a new applied science.

"Medical doctors are interested in the individual patient's welfare at all costs," Sanders says. "I would demand that from my own dr. or a medico for a member of my family. They are not necessarily the all-time evaluators of the societal and public health furnishings of their procedures. When confronted with seriously ill patients for whose condition in that location is no existing effective treatment they call up, 'Let's become ahead and exercise the experiment anyhow. Maybe it will piece of work.'"

Commencement FDA-approved gene therapy for cancer may be on its style

One frontrunner for the distinction of being the first gene therapy to win approval from the U.S. Nutrient and Drug Administration is Advexin, made by Introgen, of Austin, Texas, and which resembles Gendicine. Both utilise an adenoviral vector to deliver the p53 tumor suppressor cistron and can be used in combination with radiotherapy.

Advexin is being studied in two phase three trials that are enrolling patients with recurrent, unresectable head and cervix squamous cell carcinoma. In i trial, Advexin monotherapy is being compared to methotrexate monotherapy, the primary outcome being the effect on survival time. The other compares combined chemotherapy plus Advexin versus the aforementioned chemotherapy without Advexin, with the principal endpoint being the fourth dimension to progression.

The FDA has designated Advexin as an orphan drug, so if it's approved, Advexin could win seven years of marketing exclusivity. Advexin too is existence studied as a treatment for many other cancers.

Looking at gene therapy from the perspective of public wellness, Sanders says he's opposed to doing gene therapy experiments as well early. "Yes, we did learn some things from the death of Jesse Gelsinger [the 19-year-old who died during a gene therapy experiment at the Academy of Pennsylvania in 1999], just we didn't have to larn them in that way," he says.

Equally an case of how physicians' haste to aid patients tin lead researchers in the incorrect direction, Sanders cites a cardinal study from W. French Anderson that paved the way for the starting time human gene therapy experiments. With the business organization that a viral vector might learn the ability to replicate, it was of import to demonstrate that replication did not occur if a retrovirus was used every bit a vector. Toward this end, Anderson and colleagues injected rhesus macaques (a proxy for man subjects) with a replication-competent murine leukemia retrovirus. Because no infection developed in the macaques, the researchers concluded that murine retrovirus probably would not pose an acute health hazard in humans. The researchers attributed the lack of infection in the monkeys to neutralization of the retrovirus by complement.

However, there was a problem, Sanders says. The presence of complement implies the presence of antibodies against the retrovirus. The question of where those antibodies came from should take given the researchers pause. When some other squad of researchers did a similar experiment at a later date, they used a recombinant retroviral vector that was contaminated with a replication-competent virus. And, whereas in the start experiment the virus was introduced in vivo, in the second the virus was mixed with cells in civilisation, and the cells were reintroduced into the monkeys. This time, the monkeys developed leukemia.

"At that place was a huge amount of retroviral replication," Sanders says. The results differed, he explains, because mice and most other mammals possess a carbohydrate, galactose-blastoff (one,3)-galactose, every bit a component of glycoproteins on cell surfaces, but Onetime Globe monkeys (and humans, along with the slap-up apes) lack this carbohydrate. Yet, they have aplenty antibodies confronting information technology — from 1 to 3 percent —because alpha-galactose is found on the coat of many viruses that infect these animals. When the antibodies recognize a virus sporting this sugar, the complement cascade is triggered. That's what happened in the first experiment, creating the illusion of rubber. In the 2d experiment, with the transduction taking place in a cell culture instead of in vivo, at that place was no possibility of an immune response. As a result, the viruses produced in culture lacked the saccharide and went undetected and replicated, causing leukemia when the cells were returned to the macaques.

Ecology OF GENE THERAPY

Sanders likens gene therapy to the evolution of transgenic crops. Every bit he sees it, the major issue with transgenic plants isn't their potential to touch human health, as many critics fear, but rather the possibility that genes introduced into crops in the promise of improving agriculture might find their way into other plants with detrimental results for the whole environment. He thinks similar ecological problems use to cistron therapy. If someone receives a treatment with a recombinant virus that has the potential to replicate, can untreated individuals too exist receiving gene therapy? And if Deoxyribonucleic acid enters the germ line every bit a result of gene therapy, the effects could exist felt by future generations.

If gild supports widespread cistron therapy directed at disease prevention — in addition to treating existing affliction —Sanders wonders, who volition decide how and what resources should be allocated toward that finish?

The societal implications of cistron therapy are so profound that a cautious, step-by-step approach is warranted, Sanders says. "Cistron therapy volition become a component of 21st century medicine. There's no reason it can't work. But huge questions remain to be resolved. The history of mankind tells us that whenever you accept new technology, you lot have problems. But past now we should be intelligent plenty to conceptualize the problems that might be associated with gene therapy."

What are some of these issues? For starters, should patients who have received cistron therapy be allowed to donate blood? Probably non, in Sanders's opinion, given how niggling nosotros know at this point about long-term outcomes in gene therapy. And then at that place's the important political question of whether the gimmicky pharmaceutical industry should serve as the model for the development of gene therapy. Sanders observes that the big money generated by small-scale molecules and biologics is for treating chronic diseases, non for preventing disease. If society supports widespread gene therapy directed at disease prevention in addition to treating existing disease, who will determine how and what resources should be allocated toward that finish?

Given the genetic footing for most diseases, instead of contemplating the hereafter of gene therapy, it might exist equally interesting to wonder virtually the hereafter of factor therapy in the context of drug therapy. Right now, whether the illness is cancer or CVD, gene therapy investigations for the most role are focused on developing new treatments for high-hazard patients with astringent illness — patients beyond the point where conventional treatment is effective. Somewhen, however, conventional treatments and gene therapies will overlap. Thorny ethical and political issues will have to be addressed, but, over the long term, the future of drug therapy could be gene therapy.

Footnotes

^aneSince 1974, the RAC has reviewed technology involving recombinant Deoxyribonucleic acid, including clinical trials involving man factor transfer if direct or indirect NIH funding is provided. Since 1997, the U.South. Nutrient and Drug Administration has been the primary regulator and overseer of gene therapy trials, while the Recombinant Advisory Deoxyribonucleic acid Committee has promoted public sensation and agreement of issues surrounding gene therapy. Yet, the RAC'due south office goes beyond education, taking on powerful and public scientific, ethics, and policy advisory oversight. The RAC sends its recommendations to the FDA and local committees, and while those RAC comments formally are recommendations but, they carry great influence on the concluding actions taken by the local review committees.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564347/

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