Scientists Put CRISPR on Safer Path to Patient Treatments with New Process for Evaluating Impacts of Gene Edits that Alter Rather than “Knock Out” DNA Code

Mar 22, 2022

5 min

In new study in journal Gene Therapy, researchers at ChristianaCare’s Gene Editing Institute describe how the advance is validating the safety and efficacy of their novel approach for using CRISPR to improve lung cancer treatments


A new study from scientists at ChristianaCare’s Gene Editing Institute is advancing the safety and efficacy of using CRISPR gene editing in patient treatments by demonstrating how to identify and evaluate the broad-based biological impact of gene editing on targeted tissues, where the edits are designed to fully disable or “knock out” a specific sequence of genetic code. The work, published today in the Nature journal Gene Therapy, supports the Institute’s efforts to improve lung cancer treatments by using CRISPR to disable or alter a master regulator gene to prevent it from producing a protein that blunts the impact of chemotherapy.


“We found that when you use CRISPR, the edits sometimes end up altering rather than completely disabling the target gene, so we developed a process to gain a more complete understanding of what that means for patients,” said Eric Kmiec, Ph.D., executive director and chief scientific officer of ChristianaCare’s Gene Editing Institute and the principal author of the study.


Dr. Kmiec said that for his team’s lung cancer work, “We discovered that even when our CRISPR-based genetic manipulation did not completely disable the targeted gene, it altered it in ways that appear to make lung cancer tumors more sensitive to chemotherapy.


Validating lung cancer research using CRISPR

“We were fortunate that our strategy for using CRISPR to improve lung cancer treatments has been validated once again,” he added. “But our commitment to conducting an unbiased assessment of our approach highlights the importance of examining all potential outcomes of an attempt to use CRISPR to knock out a specific gene. Specifically, anyone developing CRISPR therapies needs to be on the lookout for edits that don’t fully knock out a section of DNA code—and evaluate the potential impacts for patients. They could be positive, as they were in our case, negative or neutral, but they need to be known.”


Much of the excitement around medical applications of CRISPR involves using the tool to disable harmful genes by editing or “knocking out” a specific sequence of DNA code. But there is increasing evidence that in the wake of a CRISPR edit, cells may remain that contain merely an altered form of the targeted code that allows the gene to continue to produce biologically active proteins.


Scientists at the Gene Editing Institute are investigating the potential of using CRISPR to disable a gene called NRF2 to alter production of the protein that protects squamous cell carcinoma lung cancer tumors from the effects of chemotherapy or radiation. They already have shown, in studies with tumor cells and in animals, that they can selectively target the NRF2 gene without affecting normal cells, where the gene confers health benefits.


In the present study they wanted to go further. They wanted to fully understand the implications of a CRISPR gene edit that allowed the NRF2 gene to retain enough DNA code to continue making a version of the protein, albeit in an altered or truncated form. The team is laying the groundwork for a clinical trial that would use CRISPR to improve the efficacy of conventional chemotherapy and radiation treatments. Dr. Kmiec said that before proceeding, he wanted his team to develop a clear process for identifying and evaluating all outcomes of CRISPR edits.

Identifying and understanding the diversity of genetic outcomes produced by CRISPR-directed gene editing has been a centerpiece of the foundational research programs established by the Gene Editing Institute.


Using CRISPR in a safe way

“We carry out experiments in an unbiased fashion, not hoping for a particular outcome, but with patient safety and efficacy serving as the true north for our scientific endeavors,” Dr. Kmiec said. “No matter what we uncover or elucidate, the insights will help both ChristianaCare and the entire field use CRISPR in a safer and more efficacious manner.”


The researchers found multiple cells where the targeted strand of DNA code in the NRF2 gene was not completely knocked out. Rather, following the CRISPR edit, cells emerged that had retained enough of the original code to continue producing a different form of the protein. Tests revealed that cancer tumor cells generating these altered proteins may be more vulnerable to chemotherapy drugs.


"For the work we are doing with NRF2, the truncated proteins generated by the CRISPR edit appear to be beneficial for making tumors more sensitive to treatment,” said lead author Kelly Banas, Ph.D. “But the key point is these proteins were clearly biologically active. And that means we needed to determine their potential impact on the safety and efficacy of using CRISPR to treat lung cancer patients.”


Dr. Banas noted that the study points to the limits of considering a CRISPR edit to be successful simply by testing for the absence of a targeted protein in its original form. She said by that standard, their edit was successful. The edited NRF2 genes were no longer producing the same protein. But she said if that’s all the ChristianaCare team had looked for, they would have missed the altered proteins coming from the NRF2 gene—and overlooked an important outcome that, in this case, strengthens the original hypothesis and experimental approach: that using CRISPR to target the NRF2 gene holds promise for improving outcomes for lung cancer patients.


Importance of due diligence

“The process we describe in this study is a template that should be followed in any effort to develop CRISPR as a medical treatment,” Dr. Kmiec said. “We’re part of a health care organization where patient safety is the top priority. We also are working at the vanguard of an exciting area of cutting-edge medicine, where a failure to conduct due diligence could cause tragic outcomes that would set back this field for decades. With this study, we have validated a process that can help this field move forward rapidly but safely.”


CRISPR stands for “clustered regularly interspaced short palindromic repeats.” It is a defense mechanism found in bacteria that can recognize and slice up the DNA of invading viruses. Scientists have learned how to modify this mechanism so it can be directed to “edit” specific sequences of DNA code.


About ChristianaCare’s Gene Editing Institute

The Gene Editing Institute, a worldwide leader in CRISPR gene editing technology and the only institute of its kind based within a community health care system, takes a patient-first approach in all its research to improve the lives of people with life-threatening disease. Since 2015, researchers at the Gene Editing Institute have been involved in several ground-breaking firsts in the field, including the development of the first CRISPR gene editing tool to allow DNA repairs outside the human cell which will rapidly speed therapies to patients and the ExACT ™pathway of single-stranded DNA repair, which increased the on-target efficacy of CRISPR and paved the way for new CRISPR breakthroughs in precise DNA edits. Its researchers created CRISPR in a Box™, the leading educational toolkit to teach gene editing, DECODR™, recognized as the most user-friendly and precise analytical tool to understand the diversity of genetic outcomes of gene editing and are currently developing a patient trial for lung cancer using CRISPR.



You might also like...

Check out some other posts from ChristianaCare

5 min

Can You Hack Your Cycle?

You’ve probably heard of – or experienced for yourself – that women crave chocolate due to PMS (premenstrual syndrome) before or even during their cycle. Some attribute this craving to a loss of riboflavin during this time. But it could also be a response to the body’s increase in hormones that contribute to cravings for sweets and fats, and chocolate has both! Ignoring those cravings isn’t going to work. However, there are ways to satisfy your body’s needs without breaking the calorie bank, overindulging in food or reaching for unhealthy options. The cycle There are essentially two phases in the menstrual cycle — follicular and luteal — which are triggered by hormonal changes. “Cycle syncing” is the process of altering diet and activity according to a woman’s naturally occurring menstrual phases each month. When you consider nutrition “cycle syncing,” which is tailoring your nutrition with your menstrual cycle throughout the month, it’s helpful to break it into four separate components: Menstruation (beginning of menstruation). Follicular. Ovulation (1st day of luteal). Luteal. What does this mean? It means this complex cycle is controlled by female hormones that cause regular bleeding (periods). Estrogen is one of the major players that helps regulate a woman’s menstrual cycle and is produced mainly by the ovaries, the same two small glands that store hundreds of thousands of tiny eggs for release over a woman’s lifetime. Eating for PMS in cycle stages Phase 1: Menstruation – “Aunt Flo” comes to visit (3-7 days) During the menstrual period, the most work happens during the first three days. This is when a woman’s flow arrives and pain onsets in the pelvis, legs, back and other areas. This is also when the most blood is lost. The cramps a woman feels serve a purpose. They are a symptom of the uterus contracting, helping to shed the endometrium (the lining of the uterus), which you may know as Aunt Flo coming to visit, also known as menstrual bleeding. Focusing on good sources of nutrition with magnesium in them may help reduce the severity of symptoms for better sleep, headaches, muscle soreness in the uterus and cramping, swelling and bloating and mood changes. Healthy fats can be useful during this phase since levels of estrogen and progesterone are typically low. We need healthy fats to help generate any kind of hormones. Look for good sources of healthy fats in seafood rich in omega-3 fatty acids, avocados, olive oil, sardines in oil, nuts and seeds, fiber, apples, berries, ground flax seed and oatmeal. Phase 2: Follicular – release the kraken! (7-10 days) Let’s face it, during the second stage of a woman’s cycle, your body is about to drop the mic! Well, not the mic per se, but your body is preparing to release an egg. That’s a lot of work. During this phase, hormones are released to signal the production of follicles on the surface of an ovary. A handful will enlarge, but only the largest and strongest follicle will ultimately release an egg during ovulation. This plays the biggest role in the length of your cycle. At the very same time, the endometrium starts to thicken in case you’re getting ready to have a baby. The last five days of this phase, plus the ovulation day (the day the egg drops), are your fertile window (baby-making time!). This is when you are most likely to become pregnant if you have sexual intercourse without using birth control. Focus on complex carbohydrates during this phase. Complex carbohydrates keep the insulin-glucagon ratio even and can help manage depression, fatigue and insomnia. Pro Tip: Between cycling, zinc can be helpful for ovulation and potentially help to support the egg quality. For women who are looking to conceive, they may want to consider using more zinc in their diet or with a supplement leading up to the follicular phase. Talk with your health care provider to see if this is a helpful option for you. Phase 3: Ovulation phase – there can be only one! (2-4 days) The three to five days leading up to ovulation and the day of ovulation itself are the most fertile. Ovulation begins on the day the egg is released from the egg follicle on the ovary. Remember when you were little and found a dandelion seedling in the grass? You grabbed it and blew on the seedlings to release them into the wind. This is similar except it’s just one egg being released. Protein is your friend during this phase. Protein will help keep your blood sugars steady, keep you feeling full and prevent cravings. Phase 4: Luteal (premenstrual) phase – winter is coming! (10-14 days) The luteal phase starts on the day the egg drops, also known as ovulation day. This happens anytime from day 7 to day 22 of a normal menstrual cycle. After the teen years and before perimenopause (the time before menopause), the luteal phase is very predictable. It normally lasts 13 to 15 days from ovulation until menstrual bleeding starts a new cycle. These two weeks are also known as the premenstrual period. It’s very common to have symptoms during all or part of the luteal phase. You may feel irritable and cranky, gain water weight and feel bloated. A day or more before your period, you may start to have pain (cramps) in your belly, back or legs. It’s normal to have less energy at this time. Don’t panic, and don’t get down on yourself for needing to rest or nap. You may also have headaches, diarrhea or constipation, nausea or dizziness. When premenstrual symptoms make your daily life difficult, you are said to have premenstrual syndrome (PMS). Good dietary sources during the luteal phase should include calcium. Calcium can help reduce the severity of PMS symptoms, including bloating, depression, pain, mood swings and food cravings. Pro tip: It may be difficult to get all the calcium necessary to help with general PMS symptoms. While we recommend getting as much calcium as possible in your diet, check with your health care provider for guidance on nutrition and supplementation. According to the American College of Obstetricians and Gynecologists, 1,200 milligrams of magnesium is usually recommended to help reduce the physical and mood symptoms of PMS. Everyone metabolizes food and supplementation differently. Results will vary. The role of iron Consider increasing iron and iron sources, especially during the luteal phase leading up to the menstruation phase. The leading cause of iron deficiency and anemia is a woman’s cycle. The more that women boost their iron stores before their menstruation cycle, the better. Look to increase iron consumption through animal sources, legumes or fortified cereals. Some women might need supplementation or other medical interventions. Sometimes, other conditions cause heavy menstrual cycles or reasons for medical intervention. Follow up with your health care provider Women may have menstrual-related conditions needing treatment beyond lifestyle changes alone. Seek care for: Irregular periods (not due to medication or breastfeeding). Heavy menstrual bleeding or severe menstrual pain. Significant depression and/or anxiety around the menstruation cycle. Menstrual cycles can have many different symptoms and associated medical conditions. Making lifestyle modifications is helpful, but if you’re experiencing severe or concerning symptoms, these should always be discussed with a health care provider.

2 min

Post-Pandemic: How Alcohol Is Harming the Health of More Women

The prevalence of alcohol-related complications has been steadily increasing over the years, but a significant surge occurred during the pandemic, particularly among women aged 40 to 64. According to a JAMA Health Forum report titled High-Acuity Alcohol-Related Complications During the COVID-19 Epidemic, there was a significant increase in severe alcohol-related complications in women and alcoholism. The majority of these, 54% to 66%, were alcohol-related liver disease; smaller numbers, 3% to 5% and 1% to 3% were attributed to alcohol-related cardiomyopathy and alcohol-related gastritis with bleeding, respectively. What the trends mean If these trends persist, the increases in alcohol-related complications will continue to harm women. There are several reasons explaining this increase. In addition to the overall increase in alcohol consumption, the pandemic exacerbated feelings of loneliness and uncertainty about the future. Additionally, lockdowns led to more time spent at home – an environment where alcohol is readily available. How alcohol affects women Women are particularly vulnerable to the adverse effects of alcohol due to differences in body structure and chemistry. They absorb more alcohol and take longer to metabolize it. This means smaller amounts of alcohol can cause more serious health issues. Also, the shift towards drinking at home, which was further made easier by convenient online shopping and home delivery during the pandemic, led to increased consumption without judgment. Addressing any substance misuse needs a multimodal approach. There are treatment options and medications that can help. It is important to wean yourself off alcohol with a doctor’s supervision, as withdrawal can be severe and dangerous. Getting help Health care providers, patients and their loved ones can all partner in enhancing education and awareness about alcohol use risks and reducing stigma in seeking treatment when needed. It’s important for women to recognize that they are not alone and that help is available. It is a difficult addiction for anyone to go through, but education and support can significantly improve outcomes.

1 min

Sports Betting: High Stakes at a Young Age

While life may feel like a game, gambling addiction is far from child’s play. Young people are particularly vulnerable and at higher risk for gambling disorders. For those still engaged in their athletic days or recently cut from the team, sports betting may be a particular draw by familiarity. The rush from gambling is not all about winning but about the excitement and thrill of the gamble itself. It can quickly become a behavioral disorder. Why are young people at risk? Young people are particularly at risk since their brains are not yet fully developed, and they indulge in more risk-taking and reward-seeking behaviors. Also, they may have fewer responsibilities and lesser accountability for their losses. The rise of smartphones has made sports betting more accessible than ever. Young people are particularly attached to their phones. It’s not mobile phone addiction but cell phone dependence coupled with the fact there is no judgment when gambling online. The anonymity can make them more likely to take the risk. Recognize red flags It is crucial to identify problem behaviors. Warning signs include: Emotional – increased anxiety and irritability, limited frustration tolerance, inability to concentrate on other activities when not gambling. Behavioral – negative effects on personal, social and work or school responsibilities. If you see or experience these signs, seek help. You or your loved one may benefit from counseling that involves cognitive behavioral therapy, which can reframe their thinking and reinforce positive behaviors over gambling.

View all posts