PNA Enables Gene Editing without Exogenous Enzymes Added

Scientists at Carnegie Mellon University and Yale University have developed a new gene editing system, consisting of synthetic, biocompatible nucleotide technology, that has cured a genetic blood disorder in living mice. The new technology, pioneered at Carnegie Mellon's Center for Nucleic Acids Science and Technology (CNAST), relies on peptide nucleic acid (PNA) molecules. Unlike the CRISPR/Cas9 gene-editing technique, this new synthetic nucleotide technology can be administered to living animals and it also significantly decreases unwanted, off-target, gene mutations. The PNA’s are fitted inside an FDA-approved nanoparticle delivery system for transportation. According to the researchers, the chemistry behind the design of the PNA molecule is innovative because it makes the PNA water-soluble and biocompatible, which means that it doesn't bind to proteins and other biomolecules in a non-specific manner, and the distinct stereochemistry of the synthetic molecule also makes it bind to DNA more easily.

The findings of this study, published in Nature Communications, are said to offer a new approach to treat genetic diseases targeting genes in hematopoietic stem cells. In their study, the researchers targeted the gene for beta-thalassemia, a blood disorder that reduces the production of hemoglobin. Beta-thalassemia is a common target for gene editing because the disease results from defective blood cells. It seems that the PNA system “tricks” the cell's normal repair machinery into chemically altering the bad gene. This is different from other gene editing strategies, (CRISPR), which involve adding an active enzyme into the cell from the outside.

"We have developed a system that uses FDA-approved nanoparticles to deliver our PNA molecule along with a donor DNA to repair a malfunctioning gene in living mice. This has not been achieved with CRISPR," said Danith Ly, professor of chemistry in Carnegie Mellon's Mellon College of Science and an expert in PNA chemistry.

CRISPR/Cas9-mediated genome editing relies on enzymes to cleave open the DNA at a target site, and it uses the cell's normal repair machinery to repair the gene. Using CRISPR, it is difficult to administer large enzymes directly to living animals, and once the enzyme is inside a cell, the enzyme may indiscriminately cut DNA at nontarget sites. As I understand it, the reliance on the enzyme and native repair systems is what leads to different efficiencies of CRISPR/Cas9 in different cell types and organisms.

The newly designed PNA molecule is designed to cleave the double-stranded DNA molecule and bind near the target site in a highly specific manner without cutting anything. PNA, "peptide nucleic acid", is a molecule with the same bases as DNA and RNA, but instead of the sugar-phosphate backbone of natural nucleic acids, the molecule has a peptide backbone, similar to proteins. This means the molecule can base-pair with standard nucleic acids, but has other properties that allow it to carry out different functions. Most notably, PNA can form triple-helix interactions with DNA, which seems to be linked to its role in gene-editing.

The lack of provided enzyme to carry out the biochemistry of this innovative gene editing technique is the really remarkable thing about this study. Gene editing as a whole has been attached to the name "CRISPR", which is only one method for gene editing, although by far the most successful.  There has been so much CRISPR-related research recently, that the fact that this gene editing technique isn’t CRISPR is noteworthy.  More proof of concept will be important before taking the new synthetic gene editing technology to patients, and if proven effective, the earliest human application of this type of technology would definitely be about a decade or more away. I think it’s awesome though, that “nanoparticle delivery”-systems are currently being developed, and it’ll be interesting to see how synthetic biocompatible techniques will shape our current understanding of the genome.

New Gene-Editing Technique Makes Strides Towards HIV Resistance

According to the Centers for Disease Control (CDC), 1,155,792 people in the United States have been diagnosed with acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV). HIV targets T-cells via the CCR5 gene receptor which serves as a channel for the virus into cells. The virus then replicates inside the T-cells, eventually killing the host cells. This destruction of T-cells ultimately results in a highly susceptible immune system.While countless research projects aspire to develop a cure for the devastating virus, an approved cure has yet to be determined. However, hopes for finding a cure remain as vast strides towards a cure have been accomplished by some promising research.

Using a new gene-editing technique, researchers for the Harvard Stem Cell Institute (HSCI) at Massachusetts General (MGH) and Boston Children's (BCH) hospitals have created an effective technique for blocking HIV from invading and destroying its subject's immune system. The researchers effectively and precisely used CRISPR-Cas gene-editing technology to edit clinically relevant genes out of human hematopoietic stem cells and T-cells. The team was able to remove the CCR5 gene receptor out of of hematopoietic stem cells and demonstrate that these cells could differentiate into functional blood cells without the CCR5 gene. This outcome suggests that gene-edited stem cells could be delivered into HIV patients by bone marrow transplantation. The procedure would result in an HIV-resistant immune system. Dr. David Scadden, co-director of HSCI, stated that the new work is "a tremendous first step in editing out what makes human cells vulnerable to HIV."

The team identified areas of caution regarding the future of the new gene-editing therapy such as unexpected complications with the new therapy and the potential difficulty involved in treating people in the areas where HIV is most prevalent.The team also believes the new therapy will be ready for human safety trials within 5 years. The new therapy will undergo animal trials, and once they are completed, the team will apply for phase I human trials.

The more I read about the advances in gene-editing techniques, the more I am humbled at how far medical technology has come in such a short time period. I am excited to follow the development of this therapy through its trials. Advances such as this will encourage hope globally regarding the devastating virus.                                                                                                                                                                                                                                                                                                                                                                                                                                                                       
 Article: http://news.harvard.edu/gazette/story/2014/11/a-promising-strategy-against-hiv/
Related Article: http://www.nejm.org/doi/pdf/10.1056/NEJMoa0802905

Potential Cure for HIV

According to the World Health Organization (WHO), approximately 0.8% of adults between the ages of 15-49 years old are living with human immunodeficiency virus (HIV) worldwide. Despite the global prevalence of the virus, there has not yet been a functional cure. However, recently an HIV-positive patient with leukemia was cured of the virus. The patient underwent total body irradiation (TBI) and a bone-marrow transplant. The bone-marrow donor had a mutation which prevents the function of the CCR5 gene. The CCR5 gene codes for a protein which allows HIV to enter human cells. The mutation the donor possessed, however, protects cells against HIV infection.

Experts believe three factors may have contributed to the cure in the Berlin patient. The first factor which may have contributed to the patient’s cure is that the removal of blood and immune cells  which occurred after irradiation killed many of the viral reservoir cells. The second contribution may have been that the CCR5 deletion mutation from the donor cells protected those cells and their progeny from HIV infection. The third contribution to the patient’s cure may have been a graft versus host reaction which resulted in the transplanted cells and their progeny attacking and eliminating the remaining HIV-positive cells.

In order to further research this potential cure for HIV, a study was conducted involving six rhesus macaques infected with simian immunodeficiency virus (SIV). The researchers harvested hematopoietic stem cells from three of the six of the macaques prior to infection with SIV and treated the macaques with antiretroviral therapy (ART). They treated the three macaques from which they collected hematopoietic stem cells with high doses of radiation. The radiation killed 94%-99% of the monkeys’ CD4-T cells. The three monkeys then received their own virus-free hematopoietic stem cells. The viral load rebounded rapidly in the three control group monkeys and in two of the monkeys who received the transplant. Due to the failure of the treatment, the researchers suggest that “the use of the CCR5 mutant donor and/or the presence of graft versus host disease played a significant role” in the cure of the Berlin patient.

Overall, this potential treatment for HIV seems to have promise and should definitely be more extensively researched. This treatment involving ART, irradiation, and a hematopoietic stem cell transplant is very complex, and there are many aspects of it which should be thoroughly examined. I found this article extremely interesting because the graft versus host disease in the Berlin patient seemed to have been a vital factor in how the patient was cured of HIV. Due to the complexity of the Berlin patient’s case, the results will be difficult to replicate; however, replication of the Berlin patient’s results has profound implications in the search for a cure for HIV.

Article: http://www.sciencedaily.com/releases/2014/09/140925141220.htm

Related Article: http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004406

Loss of Essential Blood Cell Gene Leads to Anemia

Recently in Science Daily news there has been a discovery of a new type of gene which regulates hemoglobin synthesis during red blood cell formation. These types of findings have been able to relate to treatment of human anemias and different types of mitochondrial disorders. Researchers first tested this on zebrafish. The fish were genetically screened to clone this mitchodontrial factor connected to the gene called Atpif1.



This is the gene which allows to successfully make hemoglobin. Hemoglobin is the protein in red blood cells which allows the transportation of oxygen in the blood. Atpif1 not present in the body can result into anemia. This gene also regulates enzymatic activity of ferrochelatase. This is also known as Fech, which is the terminal enzyme in heme which is a part of hemoglobin. This gene has allowed to successfully modulate the synthesis for the heme production. The deficiency of gene Atpif1 can relate to congential sideroblastic anemias. This is a disease in which the bone marrow produces ringed sideroblasts rather than healthy red blood cells.

[caption id="attachment_5739" align="aligncenter" width="512" caption="Atpif1"][/caption]

There is constant research being done in order to identify genes which can be responsible for hematopoietic stem cell development. Slowly researchers are looking into more of these works they have been doing and creating better opportunities for human congenital anemia’s. The number of visits emergency departments with anemia as the primary diagnosis has been estimated as to 5.3 million. Hopefully there will be more research done for many more successful treatments among humans.

Metastatic Melanoma in Mice Produces Complete Remission

This article was about Gene Therapy for Metastatic Melanoma in Mice. The Indiana Unversity School of Medicine introduced melanoma T cell receptors to hematopoietic stem cells of mice. The T cells are deisnged to recongnize specific proteins of melanoma. After performing the transplantation assistant Professor Touloukian reported, "We found that the transplantation of gene-modified hematopoietic stem cells results in a new host immune system and the complete elimination of tumor." This research has helped for a new clinical trial for humans which will start by late 2011.