DNA render

Gene Edits for Enhancement

The fifth installment of our blog series about gene editing focuses on gene edits and editing for research purposes. We hope you that you find it informative – please Contact Us with any comments! View the other posts in this series!

Earlier this year, a Chinese scientist reported the birth of twin girls whose genomes had been modified to silence the CCR5 gene.

Human genome: All of the genetic information needed for the embryonic development and adult function of a human being.

The birth was reported to be one of a series of human embryo experiments designed to render the offspring resistant to infection by HIV and to prove the principal that gene editing was possible — and perhaps beneficial— in human embryos.

Gene: A specific sequence of A, C, G, T units that instruct the sequence of amino acids that comprise a specific protein. Humans have 20- to 25 thousand genes

The work was not reported in a scientific format, so few scientists have had the opportunity to review the data in detail.

CCR5: A member of the C-C chemokine protein. receptor family that codes for the docking protein for the HIV virus on the surface of HIV target cells.

Several ethical concerns with this report, if true, have been raised. The gene editing was not performed to correct a known, serious medical issue in the embryos. It was performed to enhance resistance to HIV. A highly controversial idea.

CRISPR/Cas: “Clustered Regularly Interspaced Short Palindromic Repeats” is a term that describes DNA sequences in the viruses that infect bacteria. The immune system of bacteria includes a family of proteins (CRISPR-associated, Cas) that recognize CRISPR sequences and degrades them. The enzyme, Cas, needs to bind to a specific RNA sequence of 120 units, which can be synthesized synthetically, in order to degrade the DNA. These two components also function well in cell types other than bacteria, and so have become a useful tool for cutting DNA, resulting in either small deletions, or successful insertions of new synthetic DNAs. Both outcomes create an edited (mutated) gene.

But a more practical problem with the work is the possibility of “off-target” gene edits. Much research has been devoted to discover, and eliminate, the random edits that may occur at other than the gene locations being specifically targeted by the CRISPR/Cas reagents.

Gene edit: A modification of a specific sequence of A, C, G, T units that instruct the sequence of amino acids that comprise a specific protein. The edit may or may not alter the amino acid sequence and the protein.

It is these potentially deleterious unintended consequences that must be addressed in order to protect the offspring produced.

Gene Edits for Treatment of Disease

The fourth installment of our blog series about gene editing focuses on gene edits and editing for research purposes. We hope you that you find it informative – please Contact Us with any comments! View the other posts in this series!

Gene: A specific sequence of A, C, G, T units that instruct the sequence of amino acids that comprise a specific protein. Humans have 20- to 25 thousand genes

As part of our ongoing blog series about gene editing techniques and uses, the Bedford Research Foundation presents:

Gene Edits for Treatment of Disease

Most scientists have applied the CRISPR/Cas system to specific tissues or to stem cells. For example, it is theoretically possible to repair the X-chromosome mutations in liver cells so normal blood clotting factors can be produced by the liver.

CRISPR/Cas: “Clustered Regularly Interspaced Short Palindromic Repeats” is a term that describes DNA sequences in the viruses that infect bacteria. The immune system of bacteria includes a family of proteins (CRISPR-associated, Cas) that recognize CRISPR sequences and degrades them. The enzyme, Cas, needs to bind to a specific RNA sequence of 120 units, which can be synthesized synthetically, in order to degrade the DNA. These two components also function well in cell types other than bacteria, and so have become a useful tool for cutting DNA, resulting in either small deletions, or successful insertions of new synthetic DNAs. Both outcomes create an edited (mutated) gene.

Bedford Research scientists are applying the technology to edit B2M gene sequences in unfertilized eggs which are subsequently activated for stem cell derivation.

Gene edit: A modification of a specific sequence of A, C, G, T units that instruct the sequence of amino acids that comprise a specific protein. The edit may or may not alter the amino acid sequence and the protein.

But more recently other scientists have applied CRISPR/Cas technology to human embryos. Last year a Portland Oregon research team reported their efforts to repair a mutation in the gene MYBPC3 known to be associated with acute heart failure in young men. The 30-member team created embryos with sperm from a man carrying the mutated gene in half of his sperm. (It is important to note that this experiment is not possible in Massachusetts because the stem cell bill (MGLc 111L) specifically prohibits the creation of embryos for research purposes only.)

At the time of fertilization of eggs with the mutant sperm, the Oregon scientists also injected the CRISPR/Cas agents designed to home to the gene mutation and insert “normal” DNA sequences. They reported the repair was successful in some embryos, but not all. Other research teams in New York and Australia replied to the report with their own interpretations of the results and all groups agreed much more work is needed to understand how to reliably edit genes in early human embryos.

We cannot put this genie back in the bottle, but with reasoned approaches, humans can optimize the benefits and mitigate the dangers posed by gene editing. Many organizations are using this technology to find solutions for diseases, such as:

The Bedford Research Foundation is using CRISPR/Cas to further HIV prevention work using early Parthenote egg cells – More about our work is posted here.

Thank you for reading, please come back again next month for our posting on “Gene Edits for Enhancement”.

Bedford Research Foundation Fact Sheet

OUR MISSION

Bedford Research Foundation is a Massachusetts 501(c)(3) public charity and biomedical institute conducting stem cell and related research for diseases and conditions that are currently considered incurable.

WHAT WE DO

BRF conducts research in three principal areas: stem cells, prostate disease and HIV/AIDS.

Stem Cells

Advances in stem cell biology have put within reach the possibility of cures for conditions such as Parkinson’s disease, spinal cord injury, heart disease and diabetes. What is needed is a reliable source of stem cells with the broad range of developmental potential (“pluripotent”) necessary for each cell type. Although controversial, the use of human eggs may be the most efficient way to generate pluripotent stem cells. Using mouse as a model system, BRF scientists are vigorously pursuing the possibility of generating pluripotent stem cells from unfertilized eggs, encouraged by the work of BRF Trustee Dr. Jose Cibelli who has successfully derived parthenotes from monkey eggs.

Background studies of early human embryos, conducted by BRF scientists in collaboration with medical researchers at the University of Athens, Greece, revealed a potentially important role for circadian rhythms in early embryos and stem cells. Studies are ongoing to discover what circadian genes are important to cell development, and to design culture conditions to support stem cell circadian rhythms.

In parallel, BRF scientists are taking advantage of a new technology, reported in 2013, to improve the efficiency of modifying stem cells for specific treatments, such as resistance to HIV infection, and development into essential cell types, such as nerves and immune cells.

Prostate Disease

Current research is expanding diagnostic capabilities of semen specimens to identify cancer and infections of the prostate. BRF scientists have developed the first comprehensive library of bacteria types detectable in semen specimens by state-of-the-art molecular biology techniques. This work is funded primarily through The Robert C. Eyre Research Fund. Dr. Eyre, one of the BRF’s medical researchers, is a leading urologic surgeon studying the etiology, diagnosis and treatment of diseases of the genitourinary tract, including infections and cancers of the prostate, kidney and bladder.

HIV/AIDS

In 1996, BRF began a Special Program of Assisted Reproduction (SPAR) to help men infected with HIV father children without transmitting the virus to mothers or babies. The program required BRF sponsorship because of federal statutes prohibiting funding from the National Institutes of Health (NIH) for research involving fertilized human eggs. The work was possible because BRF scientists have been studying HIV transmission since the beginning of the AIDS pandemic in the early 1980’s. As of April 2015, 230 HIV-free babies have been born through SPAR.

Current work is focussed on developing stem cells resistant to HIV infection. Since HIV infects blood cells through specific receptors on the surface of the cell, if that receptor were missing, the cell would not become infected. It has been known since the beginning of the AIDS pandemic that it would be possible to cure HIV infection by transplantation of bone marrow cells resistant to HIV, but it was not until 2009 that proof of this principle was obtained when an AIDS patient with cancer underwent a bone marrow transplant to cure his cancer with bone marrow cells that were naturally resistant to HIV because they were missing the HIV receptor (“CCR5”). Approximately 1.5% of humans are missing CCR5 on the surface of their cells.

To take advantage of this possible treatment/cure for HIV, BRF scientists are studying ways to efficiently “knock-out” the gene in stem cells. The most direct approach is to knock out the gene in the eggs before they develop into stem cells, because each succeeding cell will be genetically identical to the egg. Studies are ongoing.

WHO WE ARE

Ann A. Kiessling, PhD, Director of BRF, is an expert in HIV/AIDS and stem cell biology. Dr. Kiessling has published more than 100 scientific papers and is the author of Human Embryonic Stem Cells, the first textbook on the subject. Prior to devoting herself full time to the Foundation, she directed a lab at Harvard Medical School for over 25 years. BRF is guided by its Board of Trustees, made up of medical, legal and other experienced professionals, including Professor Jose Cibelli of Michigan State University, a pioneer in stem cell research, and Chairman Alan S. Geismer, Esq., of the law firm Sugarman, Rogers, Barshak & Cohen. The BRF Ethics Advisory Board is chaired by Arthur Applbaum, Professor of Ethics and Public Policy at Harvard’s Kennedy School of Government. Carol M. Warner, Matthews Distinguished Professor of Biology at Northeastern University, is chair of the BRF Science Committee.

BRIEF HISTORY

BRF began its work in 1996. Its initial project, the Special Program of Assisted Reproduction (SPAR), was created to facilitate healthy conception for couples with a male partner infected with HIV. The first SPAR baby was born healthy and infection-free in 1998. With the help of over 200 collaborating clinics nationwide, more than 230 healthy babies have been born through SPAR.

In addition to breakthroughs in HIV, BRF’s SPAR research led to the creation of innovative methods of testing for diseases of the male genital tract, including the prostate. Success and expertise in fertility research and treatment lead to BRF scientists implementing the first human egg donor program for stem cell research in September 2000.

WHY WE DO IT

BRF exists to pursue research with the most curative potential for diseases affecting millions of people today. A 200-fold growth since its inception testifies to the excitement and importance of program goals. Through continued education and intense research efforts, BRF will change the pace of progress for diseases that currently have no cure.

A moratorium on federal funding for crucial areas of biomedical research means public charities like BRF are the only means of bridging the gap between what the government supports and what people need. Now more than ever, BRF’s innovative, cost-effective research efforts are essential to the development of life-saving procedures and cures.

HOW WE DO IT

Bedford Research Foundation is a nonprofit, 501(c)(3) Massachusetts public charity. BRF has far lower operating costs than larger institutions, meaning more research results from every donation received. BRF relies on the insightful generosity of corporations, organizations and individuals to continue its vital work.

HOW YOU CAN HELP

Support BRF by joining a network of hundreds of donors helping to shape the future of science and medicine. Financial contributions can be designated for specific programs such as stem cell or prostate research, or can go toward general research and operating costs. Donations can be given in honor or in memory of a friend, colleague or family member. Volunteers, in-kind and other support are always welcome. If you would like to help, please visit www.bedfordresearch.org.

Philanthropy Is The Key To Continued Progress

The average cost of each experiment is $90,000. Because much of our overhead is covered by fee-for-service laboratory tests, 92% of every dollar you donate goes directly toward these experiments. This innovative funding model allows Bedford Research scientists greater flexibility to move quickly in promising new research directions.

Continued Progress requires meeting our annual funding goal of $450,000 in 2018.

Gene Edits for Research

The third installment of our blog series about gene editing focuses on gene edits and editing for research purposes. Hope you that you find it informative – please Contact Us with any comments! View the other posts in this series!

Gene edit: A modification of a specific sequence of A, C, G, T units that instruct the sequence of amino acids that comprise a specific protein. The edit may or may not alter the amino acid sequence and the protein.

Early gene editing experiments were accomplished by mating individuals with different traits. Two well known examples are Mendel’s famous red peas crossed to white peas to yield pink peas (Mendel experiments summarized in this short animation: https://youtu.be/Mehz7tCxjSE), and Mr. Little’s Fancy Mice, popular in the early 1900’s, bred for coat color, formed the basis of the Jackson Laboratory’s inbred mice to study genetic diseases.

Nobel Laureate Mario Capecchi systematically studied the function of mouse genes by mutating them into silence, so called “knock-out” mice (he also spoke at the Foundation’s annual Activated Egg Symposium, in a talk titled “Gene Targeting Into the 21st Century: Mouse Models of Human Disease From Cancer to Neuropsychiatric Disorders”). This was accomplished by flooding cultures of mouse embryonic stem cells with strands of synthetic DNA that could replace the normal gene with an edited copy during DNA replication. The edited gene sequence was designed to not guide the synthesis of the normal protein. Such gene edited cells were combined with early mouse embryos, ultimately becoming part of the tissues of the mouse, including occasionally sperm and eggs. Males with gene edited sperm were mated to females with gene edited eggs to produce offspring containing two copies of the edited, non-functioning genes. Although laborious and time-consuming, this approach has yielded highly valuable information about the normal functions of thousands of genes.

In the past 20 years, other less time consuming methods of silencing genes, or increasing their expression, have been developed, all with the goal of understanding their function in health and disease.

In 2013, the most recent method for gene editing was popularized by scientists at Stanford and MIT. It is an adaptation of a naturally occurring defense mechanism that bacteria have against the viruses that invade them. Termed CRISPR/Cas, it is a complex between a protein that can cut DNA strands and a synthetic single-stranded RNA with a sequence of A, C, G, U that matches the gene being targeted (short video explanation of CRISPR here: https://youtu.be/duKV1lNiqQw). The simplicity and specificity of the system have rapidly led to a wide variety of applications among scientists world-wide.

CRISPR/Cas: “Clustered Regularly Interspaced Short Palindromic Repeats” is a term that describes DNA sequences in the viruses that infect bacteria. The immune system of bacteria includes a family of proteins (CRISPR-associated, Cas) that recognize CRISPR sequences and degrades them. The enzyme, Cas, needs to bind to a specific RNA sequence of 120 units, which can be synthesized synthetically, in order to degrade the DNA. These two components also function well in cell types other than bacteria, and so have become a useful tool for cutting DNA, resulting in either small deletions, or successful insertions of new synthetic DNAs. Both outcomes create an edited (mutated) gene. 

Such targeted DNA cuts can edit the gene sequences so they no longer code for a functioning protein, analogous to the natural CCR5 mutation, or opening the DNA strands can allow the incorporation of synthetic DNA sequences into the cut site. This raises the exciting possibility of being able to repair defective human genes. We’ll see you next month, when we’ll discuss how these research gene editing techniques may be used in the potential treatment for diseases.

New Research Program a Success in Mouse Stem Cells

Dr. Joel Lawitts microinjects CRISPR/Cas “gene editing” enzymes into mouse eggs to neutralize two genes at once: (1) the gene that leads to tissue rejection, and (2) the gene that allows HIV infection of cells. These are the first steps in generating off-the-shelf stem cells for everybody that are also resistant to HIV infection.

From the Director

The derivation of gene edited, universal, HIV-resistent human stem cells from unfertilized eggs will not be without controversy. Fortunately, we have meritorious individuals serving as our Ethics Advisory Board, our Human Subjects Committee and our Stem Cell Research Oversight Committee. Their guidance has helped us forge ahead into areas of stem cell development that larger institutions have shied away from because the work cannot be funded by our federal government. The “Dickey-Wicker Amendment” to the budget of the National Institutes of Health has been renewed annually and prohibits funds to be used for studies of unfertilized human eggs. We have for years believed unfertilized eggs (“parthenotes”) will be a broadly applicable source of “universal” human stem cells for everybody. Since human egg research MUST be privately funded, progress depends entirely on private donations.

BRF is uniquely positioned to push this exciting field forward, and we need everyone’s support!

Ann A Kiessling, PhD
Director, Bedford Research Foundation

BRF Research News

Our goal for 2017 was to improve the efficiency of a new technology, “gene editing” by CRISPR, that can precisely edit genes in eggs activated to become stem cells. BRF scientists accomplished this goal in a mouse model by developing new methods that improve the efficiency of CRISPR gene editing in mouse eggs from 10% to approximately 75%, with the added success of deriving stem cells from more than 50% of the gene edited, activated eggs.

Two genes were simultaneously targeted for editing:

(1) Just as Type “O” blood can be given to almost everyone, a “universal” stem cell could be missing the gene, B2M, responsible for the proteins on stem cells that cause immune rejection following transplantation. Such a “universal” stem cell could be transplanted into many individuals without leading to immune rejection. This is an essential step to the derivation of “off-theshelf” stem cells for everybody.

The 2017 mouse egg stem cell experiments by BRF scientists derived mouse stem cells missing B2M. This paves the way to translate the research to the derivation of universal stem cells from human eggs. Like blood banks, universal stem cell banks would be available in hospitals for acute treatments, such as heart attack, stroke and spinal cord injury.

(2) CRISPR gene editing can also mimic the natural mutation in 1% of humans that renders individuals resistant to infection by HIV, the virus that causes AIDS. The recent success in mouse eggs to eliminate the HIV receptor, CCR5, paves the way to deriving a library of universal human stem cells also resistant to HIV infection.

IF those cells can be developed into bone marrow stem cells, and IF those bone marrow stem cells will function normally, they could be utilized as a powerful treatment, perhaps a cure, for HIV disease.

Naturally Occurring Gene Edits

Continuing our series on the basics of Gene Editing, the topic of this post is inspired by the recent excitement and media coverage of CRISPR Gene Editing technology. View the other posts in this series!

No two individuals have exactly the same gene sequences because multiple sequences code for the same amino acid. This is the basis for DNA tests to prove paternity or predict ancestry. Most of the gene variations do not change the proteins they code for, but some do, such as genes for eye and hair color and height (for a quick recap of genes, check out this video: https://youtu.be/5MQdXjRPHmQ).

Gene edit: A modification of a specific sequence of A, C, G, T units that instruct the sequence of amino acids that comprise a specific protein. The edit may or may not alter the amino acid sequence and the protein.

Therefore, fertilization of an egg, pollination of a flower, introduce gene edits in the offspring because of variations in the gene sequences of the two cells uniting.

Human genome: All of the genetic information needed for the embryonic development and adult function of a human being.

Still other gene edits occur because of “transposable elements,” first described in corn by Barbara McClintock (1), Nobel Laureate in 1983. Such “transposable elements” are common in all life forms, approximately 45% of the human genome is transposable elements and their location in individual genomes is highly variable (more on Barbara McClintock and transposons here: https://youtu.be/91vR-FKBMT4).

Chromosome: a long string of genes attached end to end and then folded with proteins in a specific way.

The most well-studied gene edits in humans are those that cause cancer, such as the breast cancer gene, BRCA, on chromosome 13. It codes an important enzyme in DNA repair. A mutation that results in a “frame shift,” as described above results in no BRCA protein expression. Hence, its function to repair spontaneously occurring DNA mutations is inhibited, resulting in cells containing mutated DNA that lack the controls that limit cell multiplication, leading to uncontrolled cell expansion, the definition of cancer.

Gene: A specific sequence of A, C, G, T units that instruct the sequence of amino acids that comprise a specific protein. Humans have 20- to 25 thousand genes

A more recently studied naturally occurring gene edit is the 32 gene unit deletion in CCR5 on chromosome 3. The mutation results in loss of CCR5 protein on the surface of HIV target cells, rendering them resistant to HIV attachment and infection. This mutation naturally occurs in approximately 1.5% of humans (here is a good illustration of this mutation, just ignore the quiz question at the end: https://youtu.be/0PtBQoKD6uk)

Thank you for reading, next month we’ll be discussing more on gene edits, specifically gene edits for research.

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Federally funded research institutions around the country are being affected by the federal government shutdown, but Bedford Research never has to worry about this since we are funded by private donations! 94% of every dollar you donate goes directly toward our research, giving Bedford Research scientists greater flexibility to move the work quickly in promising new directions.

Please become a supporter and help us do more experiments this year.