Fragment of AIDS virus may be used to deliver therapeutic proteins to cells

September 03, 1999



September 3, 1999
-- A team of Howard Hughes Medical Institute (HHMI) researchers has successfully used a piece of the AIDS virus to deliver a fully functional protein inside a cell, creating a new way to deliver therapeutic proteins inside the body.

"This demonstration means two things," said Steven Dowdy, an HHMI investigator at Washington University School of Medicine in St. Louis, who developed the technique with postdoctoral fellows Steven R. Schwarze, Alan Ho and Adamina Vocero-Akbani. "First, it introduces a new way to study diseases and do experiments that no one has ever been able to do before. And second, it has the potential to open a new field called protein therapy."

Damage to proteins causes many human diseases, including cancer and genetically inherited disorders as sickle-cell anemia and phenylketonuria. Inserting a working version of a damaged protein into affected cells could be a promising therapy for such illnesses. Until now, however, researchers had been unable to coax bulky biomolecules through the densely packed fatty molecules of the cell membrane.

In the September 3, 1999, issue of the journal Science, Dowdy and his colleagues report that their way around this obstacle is to use a fragment of a protein from the human immunodeficiency virus (HIV). When hooked to a large protein, this harmless piece of HIV broaches, or transduces, the cell membrane.

"We call this transduction the parting of the Red Sea," said Dowdy. "It's as if the HIV fragment interacts somehow with the lipid bilayer of the membrane, opens it up, inserts the protein, and then seals it back up. We can't see anything leaking out from the cell."

The fragment used by Dowdy's team comes from the HIV protein TAT, which the virus normally uses to facilitate gene transcription, not to infect host cells. That the TAT fragment can penetrate a cell at all is a biochemical quirk, says Dowdy, which was first reported by researchers in 1988. As a result, it is unlikely that the HIV fragment poses any danger of triggering disease, he said.

To demonstrate that the technique works, the HHMI team hooked the 11-amino-acid TAT fragment to a peptide tagged with a compound called fluorescein that emits a green glow when illuminated by fluorescent light. The luminous peptide-fluorescein compound is four times the size of the largest compound that can enter cells naturally. Nevertheless, cells in the spleen, liver, and muscle glowed green just 20 minutes after Dowdy's team injected the proteins into the abdomens of mice.

Even the brain tissue had a solid green color, indicating that the injected proteins were able to cross the blood-brain barrier. The blood-brain barrier is a layer of tightly packed cells designed to keep most large molecules--including fluorescein--out of the central nervous system.

In a second experiment designed to see whether the technique delivers functional proteins, Dowdy's group used the TAT fragment to attempt to ferry an even larger protein--an enzyme called beta-galactosidase--into mouse cells. Beta-galactosidase helps break down complex sugars, including a stain called X-Gal that turns blue when it is broken down by beta-galactosidase.

"We held up the tissue samples from the mice and said 'Wow,'" says Dowdy. "All of the tissues were blue, indicating that the enzyme was working inside the cells."

Dowdy's group was also surprised by the rapidity of the transduction. "The evidence suggests the cells are transduced within an hour, and probably within 30 minutes," explained Dowdy. Crossing the blood-brain barrier seemed to take longer, but even this difficult task was complete within four hours.

In designing drugs, pharmaceutical companies must consider what they call the "bioavailability wall." Any therapeutic agent too large or too attracted to water cannot penetrate the fat-based cell membrane.

The limits imposed by the bioavailability wall are stringent. For example, the cell membrane will not transduce proteins larger than about six amino acids. Yet even small human proteins contain about 150 amino acids. Beta-galactosidase contains over 1,000 amino acids and is nearly 200 times the size limit set by the bioavailability wall.

A molecular oncologist, Dowdy plans to use the technique to study cancer. He is particularly interested in two tumor-suppressor proteins, p16 and RB, whose genetic pathway is mutated in almost 90 percent of cancers.

"There are already knockout mice that are missing genes for p16 and RB, and we know these mice are predisposed to develop early cancers," he said. "But now we can transduce those tumor suppressor proteins back into mice and then withdraw the proteins at various times to find out when they are really critical. We'll be able to ask very specific questions about the earliest steps that lead to cancer."
-end-


Howard Hughes Medical Institute

Related HIV Articles from Brightsurf:

BEAT-HIV Delaney collaboratory issues recommendations measuring persistent HIV reservoirs
Spearheaded by Wistar scientists, top worldwide HIV researchers from the BEAT-HIV Martin Delaney Collaboratory to Cure HIV-1 Infection by Combination Immunotherapy (BEAT-HIV Collaboratory) compiled the first comprehensive set of recommendations on how to best measure the size of persistent HIV reservoirs during cure-directed clinical studies.

The Lancet HIV: Study suggests a second patient has been cured of HIV
A study of the second HIV patient to undergo successful stem cell transplantation from donors with a HIV-resistant gene, finds that there was no active viral infection in the patient's blood 30 months after they stopped anti-retroviral therapy, according to a case report published in The Lancet HIV journal and presented at CROI (Conference on Retroviruses and Opportunistic Infections).

Children with HIV score below HIV-negative peers in cognitive, motor function tests
Children who acquired HIV in utero or during birth or breastfeeding did not perform as well as their peers who do not have HIV on tests measuring cognitive ability, motor function and attention, according to a report published online today in Clinical Infectious Diseases.

Efforts to end the HIV epidemic must not ignore people already living with HIV
Efforts to prevent new HIV transmissions in the US must be accompanied by addressing HIV-associated comorbidities to improve the health of people already living with HIV, NIH experts assert in the third of a series of JAMA commentaries.

The Lancet HIV: Severe anti-LGBT legislations associated with lower testing and awareness of HIV in African countries
This first systematic review to investigate HIV testing, treatment and viral suppression in men who have sex with men in Africa finds that among the most recent studies (conducted after 2011) only half of men have been tested for HIV in the past 12 months.

The Lancet HIV: Tenfold increase in number of adolescents on HIV treatment in South Africa since 2010, but many still untreated
A new study of more than 700,000 one to 19-year olds being treated for HIV infection suggests a ten-fold increase in the number of adolescents aged 15 to 19 receiving HIV treatment in South Africa, according to results published in The Lancet HIV journal.

Starting HIV treatment in ERs may be key to ending HIV spread worldwide
In a follow-up study conducted in South Africa, Johns Hopkins Medicine researchers say they have evidence that hospital emergency departments (EDs) worldwide may be key strategic settings for curbing the spread of HIV infections in hard-to-reach populations if the EDs jump-start treatment and case management as well as diagnosis of the disease.

NIH HIV experts prioritize research to achieve sustained ART-free HIV remission
Achieving sustained remission of HIV without life-long antiretroviral therapy (ART) is a top HIV research priority, according to a new commentary in JAMA by experts at the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health.

The Lancet HIV: PrEP implementation is associated with a rapid decline in new HIV infections
Study from Australia is the first to evaluate a population-level roll-out of pre-exposure prophylaxis (PrEP) in men who have sex with men.

Researchers date 'hibernating' HIV strains, advancing BC's leadership in HIV cure research
Researchers have developed a novel way for dating 'hibernating' HIV strains, in an advancement for HIV cure research.

Read More: HIV News and HIV Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.