Gene therapy using lipid based nanoparticles

Lipid nanoparticles (SLNs and NLCs) are regarded as highly promising systems for delivering nucleic acids in gene therapy. Literature review by researchers at PharmaNanoGene describes these systems and their main advantages in gene therapy, such as their capacity to protect the gene material against degradation, to facilitate cell and nucleus internalization and to boost the transfection process.


View of lipid nanoparticles.
View of lipid nanoparticles. Credit: UPV/EHU

“At PharmaNanoGene we are working on the design and evaluation of SLNs for treating some of these diseases using gene therapy. We are studying the relationship between formulation factors and the processes involving the intracellular internalisation and disposition of the genetic material that condition the effectiveness of the vectors and which is essential in the optimisation process, and for the first time we have demonstrated the capacity of SLNs to induce the synthesis of a protein following their intravenous administration in mice,” they stressed.

The publication also includes other pieces of work by this University of the Basque Country research group on the application of SLNs in the treatment of rare diseases, such as chromosome-X-linked juvenile retinoschisis, a disorder in which the retina becomes destructured due to a deficiency in the protein retinoschisin.

 “One of the main achievements of our studies in this field has been to demonstrate, also for the first time, the capacity of a non-viral vector to transfect the retina of animals lacking the gene that encodes this protein and partially restore its structure, showing than non-viral gene therapy is a viable, promising therapeutic tool for treating degenerative disorders of the retina,” specified the researchers.
The application of SLNs for treating Fabry disease, a serious, multi-system metabolic disorder of a hereditary nature, has also been studied at PharmaNanoGene.

 “This is a monogenic disease linked to the X-chromosome which is caused by various gene mutations in the gene that encodes the galactosidase A enzyme. In cell models of this disease we have demonstrated the capacity of SLNs to induce the synthesis of galactosidase A enzyme”. They have also reviewed the application of lipid nanoparticles to the treatment of infectious diseases: “Our work in this field shows that SLNs with RNA interference are capable of inhibiting a replicon of the hepatitis C virus in vitro, which was used as proof-of-concept of the use of SLN-based vectors as a new therapeutic strategy for treating this infection and others related to it”.

Citation: Ana del Pozo-Rodríguez, María Ángeles Solinís, Alicia Rodríguez-Gascón, Applications of lipid nanoparticles in gene therapy, European Journal of Pharmaceutics and Biopharmaceutics, Volume 109, December 2016, Pages 184-193.
DOI: 10.1016/j.ejpb.2016.10.016.
Adapted from press release by University of the Basque Country.

Making T-Cell receptor gene therapy safer by domain swapping

The human body produces T cells to recognize and fight disease. Each T cell has a unique T cell receptor (or TCR) on its surface that surveils small fragments of proteins presented by other cells. Upon detecting evidence of cancer or infection, a subset of T cells binds the diseased cells and orchestrates their elimination. When tumors and infections cannot be eradicated naturally, researchers employ immunotherapies to boost the immune system’s effectiveness.

By inserting genes encoding a tumor-specific T cell receptor (TCR) into a patient’s T cells, researchers can engineer a large population of T cells to target tumor cells. This approach, called T cell receptor gene therapy, has yielded clinical successes where conventional cancer treatments have failed. However, T cell receptor gene therapy is not without risk. The introduced receptor can become tangled with the resident receptor in each engineered T cell, causing some of these cells to attack healthy cells. A new technique developed by Caltech researchers prevents this from happening, increasing the safety of T cell receptor gene therapy.

The technique, called domain swapping, was developed in the laboratory of David Baltimore, president emeritus and the Robert Andrews Millikan Professor of Biology. A paper describing the findings appears in the November 8 issue of the journal eLife.

The specificity of the T cell receptor (TCR) in each T cell results from the pairing of two protein chains–called an alpha chain and a beta chain–each of which has constant domains (shared between all TCRs) and variable domains (unique to each T cell). Normally, each T cell encodes only one alpha chain and one beta chain, which pair to form a single TCR. In T cell receptor gene therapy, the introduction of genes encoding a tumor-reactive TCR results in T cells that express two alpha chains and two beta chains, with four possible pairings. This non-physiological situation poses a risk of autoimmunity.

The group’s solution was to generate hybrid genes encoding T cell receptor (TCR) chains with their alpha and beta constant domains swapped in a compensatory fashion. When correctly paired, these domain-swapped TCRs retain all of the domains necessary to function. Indeed, the group found that domain-swapped TCRs and unmodified TCRs both function in human T cells, and they prevented tumor growth in mice to a similar extent. However, whereas unmodified TCRs mispaired with resident TCR chains in both mouse and human T cells, and caused autoimmunity in mice, domain-swapped TCRs did not.

In addition to preventing mispairing, domain-swapped TCRs highlight a surprising robustness to the function of the TCR complex. The Caltech group teamed with Mike Kuhns at the University of Arizona to determine that domain-swapped TCRs assemble in a similar manner to unmodified TCRs despite significant structural rearrangement of the constituent protein chains. Domain-swapped TCRs may be useful tools for further study of the structure and function of the TCR complex.

Finally, in collaboration with Wolfgang Uckert at the Max Delbrück Center for Molecular Medicine in Berlin, the researchers showed that domain-swapped TCRs were expressed at higher levels on the T cell surface when the resident TCR genes were silenced.

“Our paper focuses on the increased safety afforded by domain-swapping, but combining these two solutions may result in a therapy with improved safety and efficacy compared to current practice,” Bethune says.

Citation: Bethune, Michael T., Marvin H. Gee, Mario Bunse, Mark S. Lee, Eric H. Gschweng, Meghana S. Pagadala, Jing Zhou et al. “Domain-swapped T cell receptors improve the safety of TCR gene therapy.” eLife 5 (2016): e19095.
DOI: http://dx.doi.org/10.7554/eLife.19095
Research funding: National Institutes of Health, Prostate Cancer Foundation, Jane Coffin Childs Postdoctoral Fellowship, Pew Charitable Trusts.
Adapted from press release by California Institute of Technology.

Gene therapy with Alipogene Tiparvovec (Glybera) for Lipoprotein Lipase Deficiency shows promise

Over a 6-year period, patients with the genetic disease lipoprotein lipase deficiency (LPLD) who received a single gene therapy treatment of alipogene tiparvovec had a marked reduction in the severity and frequency of pancreatitis. No cases of severe pancreatitis and only one admission to the intensive care unit for an lipoprotein lipase deficiency related abdominal event were reported in the study published in Journal Human Gene Therapy.

The researchers assessed lipoprotein lipase deficiency related acute abdominal events that required hospital care in a small group of patients treated with a single dose of the gene therapy product Glybera®. The results support an association between gene therapy for this rare genetic disease and overall reductions in healthcare costs and resource utilization.

“As gene therapy moves forward into the mainstream of medicine, it will be critical to define the evidence of its benefit to patients with specific diseases,” says Editor-in-Chief Terence R. Flotte, MD, Celia and Isaac Haidak Professor of Medical Education and Dean, Provost, and Executive Deputy Chancellor, University of Massachusetts Medical School, Worcester, MA. “This work with alipogene tiparvovec provides an evidence-based guideline for its use in patients with lipoprotein lipase deficiency going forward.”

Citation: Long-Term Retrospective Analysis of Gene Therapy with Alipogene Tiparvovec and Its Effect on Lipoprotein Lipase Deficiency-Induced Pancreatitis.
Gaudet Daniel, Stroes Erik S., Méthot Julie, Brisson Diane, Tremblay Karine, Bernelot Moens Sophie J., Iotti Giorgio, Rastelletti Irene, Ardigo Diego, Corzo Deyanira, Meyer Christian, Andersen Marc, Ruszniewski Philippe, Deakin Mark, and Bruno Marco J. Human Gene Therapy 2016 pp: hum.2015.158
DOI: http://dx.doi.org/10.1089/hum.2015.158
Adapted from press release by Mary Ann Liebert, Inc., publishers

Pancreatitis Reduced by Nearly 50% After Gene Therapy to Treat Lipoprotein Lipase Deficiency

Researchers study foamy retrovirus as a safe gene therapy vector

A Washington State University researcher has developed a way to reduce the development of cancer cells that are an infrequent but dangerous byproduct of gene therapy.

Grant Trobridge, an associate professor of pharmaceutical sciences, has altered the way a virus carries a beneficial gene to its target cell. The modified viral vectors reduce the risk of cancer and can be used for many blood diseases.

Trobridge and his team report their development in Scientific Reports, an online open-access journal produced by the Nature Publishing Group. The team is translating their findings into a stem cell gene therapy to target a life-threatening immunodeficiency in newborns called SCID-X1, also known as “Boy in the Bubble Syndrome.”

Gene therapy holds potential for treating genetic diseases by replacing defective genes with repaired ones. It has shown promise in clinical trials but has also been set back by difficulties delivering genes, getting them to work for a long time and safety issues. A joint French and English trial, for example, successfully treated 17 out of 20 patients with SCID-X1 only to see five of them develop leukemia.

Trobridge and his colleagues are using a vector developed from a foamy retrovirus, so named because it appears to foam in certain situations. Unlike other retroviruses, foamy retrovirus don’t normally infect humans. They also are less prone to activate nearby genes, including genes that might cause cancer.

Retroviruses are a natural choice for gene therapy because they work by inserting their genes into a host’s genome.

With an eye toward making the vector safer, the Trobridge team altered it to change how it interacts with a target stem cell so it would insert itself into safer parts of the genome. They found that it integrated less often near potential cancer-causing genes.

“Our goal is to develop a safe and effective therapy for SCID-X patients and their families,” said Trobridge. “We’ve started to translate this in collaboration with other scientists and medical doctors into the clinic.” He predicted that the therapy could be ready for clinical trials within five years.

Citation: Hocum, Jonah D., Ian Linde, Dustin T. Rae, Casey P. Collins, Lindsay K. Matern, and Grant D. Trobridge. “Retargeted Foamy Virus Vectors Integrate Less Frequently Near Proto-Oncogenes.”
Nature Scientific Reports
DOI: http://dx.doi.org/10.1038/srep36610
Research funding: NIH/National Institute of Allergy and Infectious Diseases
Adapted from press release by Washington State University 

Biosafety studies of Hematopoietic Stem Cell Gene Therapy for Mucopolysaccharidosis I shows promise for human trials

Extensive biosafety studies of hematopoietic stem cell (HSC) gene therapy, intended to replace a protein that patients with the inherited disease mucopolysaccaridosis I (MPS I) cannot produce, support clinical testing of the stem cell-based gene addition approach in MPS I patients. Evidence derived from these studies not only indicates that the HSC gene therapy is safe and well tolerated in mice, but also that it can produce sufficient amounts of the missing protein to affect MPS I without harming a patient’s hematopoietic stem cells, according to an article in Human Gene Therapy.

The article entitled “Preclinical Testing of the Safety and Tolerability of Lentiviral Vector-Mediated Above-Normal Alpha-L-Iduronidase Expression in Murine and Human Hematopoietic Cells Using Toxicology and Biodistribution Good Laboratory Practice Studies  is part of a special joint issue on stem cell gene therapy in Human Gene Therapy and Stem Cells & Development guest edited by Luigi Naldini, MD, Scientific Director, San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.

Ilaria Visigalli, Stefania Delai, Alessandra Biffi, Luigi Naldini and colleagues from San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, and Vita Salute San Raffaele University (Milan, Italy), Glaxo Smith Kline R&D (U.K.), Sanofi (Montpellier, France), and Royal Manchester Children’s Hospital (Manchester, U.K.), describe the lentiviral vector-based gene therapy approach they developed to deliver normal copies of the alpha-iduronidase (IDUA) gene, which contains a mutation in patients with MPS I, to HSCs. They assessed the safety of the HSC gene therapy method by studying the effects of IDUA gene transfer and production of the enzyme on human and mouse HSCs, and followed the modified HSCs and their progeny in treated mice.

“Members of this group have previously shown that lentiviral gene transfer into hematopoietic stem cells can serve as a platform for curative gene therapy of genetic diseases,” says Editor-in-Chief Terence R. Flotte, MD, Celia and Isaac Haidak Professor of Medical Education and Dean, Provost, and Executive Deputy Chancellor, University of Massachusetts Medical School, Worcester, MA. “This study sets the stage for a pivotal clinical trial to determine whether MPS I patients may also be successfully treated with this approach.”

Publication: Preclinical Testing of the Safety and Tolerability of Lentiviral Vector–Mediated Above-Normal Alpha-L-Iduronidase Expression in Murine and Human Hematopoietic Cells Using Toxicology and Biodistribution Good Laboratory Practice Studies.
http://online.liebertpub.com/doi/full/10.1089/hum.2016.068

Press release by Mary Ann Liebert, Inc., Publishers

Gene therapy for Cystic fibrosis updates

Two new studies from the University of Iowa suggest that gene therapy may be a viable approach for treating or preventing lung disease caused by cystic fibrosis (CF).

Working with CF pigs, the researchers, based in the UI Pappajohn Biomedical Institute (PBI), have shown that two different virus-based vectors can restore a working version of a protein—the cystic fibrosis transmembrane conductance regulator (CFTR)—that is faulty in CF to the pigs’ airway cells. Moreover, this gene replacement normalizes important aspects of the lung biology and improves the ability of airway secretions to kill bacteria.

“This is an important proof of principle for the idea that gene therapy for CF could work because we used an animal model that we know develops lung disease like people,” says Paul McCray Jr., professor of pediatrics in the UI Carver College of Medicine and principal investigator for one of the studies, both of which were published Sept. 8 in the journal JCI Insight. “In our short-term experiments we saw evidence of correction of some of the known problems of CF, including salt movement across the cell membrane, the pH (acidity) of the airway surface liquid, and the ability of respiratory secretions to kill bacteria. This shows that the gene therapy has an effect that appears to be therapeutically relevant.”

In the new studies, two teams tested two different gene therapy strategies to get functional CFTR into the airway cells of CF pigs. One group, led by McCray and Patrick Sinn, UI research associate professor of pediatrics and director of the UI Viral Vector Core, focused on a lentivirus. This type of virus has been successfully and safely used as a gene therapy vector for patients with rare immune diseases. A major advantage of lentiviruses is the delivered gene is directly incorporated (integrated) in to the cell’s genome, meaning the fix is permanent. However, it is challenging to produce large quantities of lentivirus, and this virus has not yet been tested for safety in human lungs.

The other research team, led by Zabner and David Schaffer at the University of California, Berkeley, focused on the adeno-associated virus AAV2. AAVs are safe for use in humans, including human lungs, and relatively easy to produce in large quantities. Genes delivered by AAV vectors are not permanently incorporated into the cell’s genome, but expression of the gene is often long-lived. An important aspect of the AAV study was the molecular customization of the virus such that it efficiently targeted pig airway cells. Zabner and Schaffer created an AAV2 virus with five mutations that was 240 times more efficient than AAV2 at infecting pig airway cells. The team previously used the same directed evolution strategy to create an AAV virus that preferentially infects human airway cells.

The researchers showed that both gene therapies restored chloride currents in pig airway cells, indicating that both vectors delivered working CFTR to the correct location in the airways. Both approaches also increased the pH and the bacterial-killing ability of the pigs’ airway secretions.

The vectors the UI researchers are developing could also have uses beyond CF gene therapy as multifaceted gene-delivery tools.

The current studies resulted from highly collaborative science among teams from the UI, the University of California, Berkeley, and Wright State University in Ohio, as well as among multiple departments within the UI Carver College of Medicine. The two lead authors of the studies are both UI graduate students: Ashley Cooney, in the microbiology program, and Benjamin Steines, in the molecular and cellular biology program. The research was funded in part by grants from the National Heart, Lung, and Blood Institute and the National Institute for Diabetes and Digestive and Kidney Diseases, both part of the National Institutes of Health, and the Cystic Fibrosis Foundation.

Press release from University of Iowa