RARE Daily

Using Directed Evolution to Develop New Vectors for Genetic Medicines

May 2, 2024

Much of the challenge of developing genetic medicines lies in having the right vector to deliver the therapy to the cells within the body where they need to go.  4D Molecular Therapeutics has developed platform technology that generates large numbers of genetically diverse, synthetic adeno-associated viral vectors that have desired characteristics using a process known as directed evolution. It is using these vectors to build a pipeline of genetic medicines across a broad set of conditions. We spoke to Alan Cohen, senior vice president of clinical development and therapeutic area head of pulmonology for 4DMT, about the limitations of existing vectors for genetic medicines, 4DMT’s directed evolution platform technology, and its programs in cystic fibrosis and Fabry disease.

Daniel Levine: Alan, thanks for joining us.

Alan Cohen: Danny, nice to get a chance to speak with you as well. Thanks for having me.

Daniel Levine: We’re going to talk about next generation gene therapy vectors, 4D Molecular Therapeutics, and its programs in cystic fibrosis and Fabry disease. Let’s start with vectors though, which are what’s used to package and carry a gene therapy to its targets. Many gene therapies today are using adeno-associated viral vectors. What makes them a popular choice?

Alan Cohen: Yeah, Danny. That’s correct. In fact, adeno-associated viral vectors or AAVs are the leading platform for the delivery of in vitro gene therapy for a wide range of rare and even common diseases. They offer several advantages compared to other viral vectors, and unlike retroviruses for example, they don’t integrate into the host genome and that reduces the risk of insertional mutagenesis. In addition, they can transfect both dividing and non-dividing quiescent targeted cells, allowing gene delivery to deliver to a highly diversified range of cell types, and using modern vector engineering techniques, they can be modified to efficiently target specific cell types and even organs. One such technique is directed evolution, harnessing the power of natural selection to yield AAV variants with clinically desirable characteristics, and at 4D, we’re employing directed evolution to invent vectors for targeted delivery to specific tissues via clinically optimal routes including the lung via aerosolization—we’ll talk a little bit about more of that later, the heart systemically or intravenously, and intravitreal through injections in the eye, and also in the future we’re looking to direct them towards the central nervous system.

Daniel Levine: Well, how constrained are we today in developing gene therapies by the existing vectors we have available? What are their limitations?

Alan Cohen: So, with respect to conventional AAV vectors, the chief limitations include the inability to efficiently target specific tissues and organs, susceptibility to neutralization by preformed antibodies, and a limited payload capacity. And in certain cases, the limited transduction efficiency requires the use of much higher doses and that of course would increase the risk of toxicity. At 4D, we’re focusing on developing vectors and transgene payloads that overcome these limitations. As previously mentioned, we’ve employed directed evolution to invent vectors with tropism for specific cell types and enhanced immune invasive capacity. In addition, we’re applying genetic engineering techniques to optimize transgene payloads and overcome the limitations related to genetic capacity.

Daniel Levine: 4DMT is creating vectors that are synthetic, they’re not found in nature. You’ve mentioned directed evolution. Perhaps you can explain the concept of directed evolution. What is 4DMT actually doing to create new vectors?

Alan Cohen: Yeah, that’s a great question Danny, and if you think about it, it’s not likely that nature intended viruses like adeno-associated viral vectors to service therapeutic vectors, but we can exploit their cell transduction capacities to deliver transgenes to cells. Directed evolution was an appropriately Nobel prize winning technology that harnesses the power of natural selection to create novel variants with specific properties by creating these massive genetic diversity and introducing selective pressure that directs the evolutionary process. It’s one of the most powerful tools for engineering AAV capsids to deliver therapeutic transgenes to targeted cells.

Daniel Levine: Well, how does the platform work?

Alan Cohen: So, our proprietary vector discovery platform, which we call the Therapeutic Vector Evolution or TVE, uses a high throughput genetic engineering platform harnessing the power of directed evolution to create customized or bespoke vectors for targeted delivery of therapeutic transgenes. TVE simulates natural evolution by introducing massive genetic diversity and applying selective pressures to yield viral capsids with novel and clinically desirable characteristics. The process actually begins with the creation of what we refer to as a target vector profile that identifies the specific vector characteristics required for the intended clinical application. A pooled library of about a billion unique synthetic variant AAV capsid sequences is then packaged in cells to produce DNA containing capsids. The resulting capsid library is then administered to non-human primates. Viral genomes are then recovered, repackaged, and readministered, and the process is repeated during iterative rounds of re-diversification and competitive selection with the aim of identifying the fittest variant for the intended evolution. The key advantage to 4D’s approach is the use of non-human primates to conduct the screening and isolation step to filter down to the dominant variant of choice to proceed to characterization, preclinical studies, and ultimately human clinical trials and commercialization ultimately of a novel genetic medicine. The beauty of this process is we don’t need to be smarter than nature. We let all the multifactorial impacts of AAV in the body, both known and unknown, play out within the process itself to select the best vector out of the many billions of possibilities. We then can use the learnings on each round of selection to continuously improve the platform process by including things like machine learning and AI techniques, re-diversification, and even adding human IVIG to select out for preexisting antibody resistance.

Daniel Levine: How random is the process? Are you just creating so many variants that you find the ones with the properties you want or can you actually control for the properties you’re seeking in generating these?

Alan Cohen: Danny, that’s a great question and as you can imagine, the rate of natural mutation is usually insufficient for generating the kind of genetic diversity required for efficient laboratory-based directed evolution. So therefore, it’s necessary to enhance the process of genetic diversification to increase the sampling of mutations. A wide range of techniques to achieve this are now available and each have their own advantages and limitations. Simply they can be classified as either random or rational mutagenesis, although certain techniques combine aspects of both. In random mutagenesis, no specific sequence positions are targeted, and this approach is particularly useful for directed evolution of proteins where there isn’t enough structure/function information available to determine which residues to diversify or when the property to be evolved cannot be easily attributed to a few specific positions. Unlike random mutagenesis, rational mutagenesis focuses on mutating only a limited number of positions in the target sequence, which must be determined based on prior knowledge. These could include things like capsid structure, multiple sequence alignments, biochemical data, and computer-based predictions. We can customize this process by selecting for specific qualities such as enhanced tropism for heart muscle cells or cardiomyocytes in a life-limiting rare disorder like Fabry’s disease, or enhanced delivery to airway epithelium and basement membrane cells in the lung for the treatment of pulmonary conditions such as cystic fibrosis or alpha-1 antitrypsin deficiency. Over the past few decades, directed evolution has demonstrated an enormous potential to help us develop variants of biomolecules with novel or enhanced properties. Many cases of successful application of directed evolution have been possible thanks to the development of new techniques for library generation and variant identification such as what we’re doing here at 4D.

Daniel Levine: How do you determine the most appropriate vectors for a specific therapy once you’ve generated these large libraries, or are you looking for its ability just to target a specific cell type or organ, or are there other qualities you seek like the size of the cargo it can carry or other things like that?

Alan Cohen: Yeah, Danny, it’s a great question. The capsid libraries, as I mentioned a moment ago, are initially administered to non-human primates and subjected to these iterative rounds or re-diversification and competitive selection, and the point is to identify the fittest variant for the desired clinical application. This again goes back to the beauty of our process. Once we’ve established diverse libraries, we go to the whiteboard and we ask ourselves a question like what tissues or diseases have the most unmet medical need and that could be addressed by an AAV genetic medicine. At 4D, we determine this step that the retina, the lung, the heart, and even the central nervous system were some of the targetable disease areas with the most unmet medical need and each with unique routes of administration we could optimize. Then we conduct these highly customized selections of each tissue type in a non-human primate, and we ended up coming up with our three lead vectors, R100 for intravitreal delivery to the retina, A101 for aerosolized delivery to the lung, and C102 for IV systemic delivery targeting the heart or cardiomyocytes. We’re currently also in early stages of vector development for targeting the central nervous system. The beauty of these vectors once discovered and characterized is that they’re modular, so once we’ve proven that they have unique and superior properties to conventional AAVs, we can leverage them and swap out the payloads for different diseases in the same tissues, and we gain enormous efficiencies this way since the majority, or 90 plus percent, of manufacturing is the same for the second time around, and the FDA often requires fewer large animal studies for each new product candidate using the same vector. This played out, for example in our R100 base portfolio where 4D-150, which we’re currently targeting for wet AMD, took approximately half the time to develop pre-clinically than 4D1110, our first R100 based product candidate for a retinal disease called CHM or choroideremia, which is a blinding and currently untreatable X-linked inherited retinal disease.

Daniel Levine: Let’s get into some of the specific programs you’re pursuing. The first I wanted to ask you about is the one for cystic fibrosis. For listeners not familiar with the condition, what is it?

Alan Cohen: Yeah, Danny, I’m glad you’re asking, and as a pediatric pulmonologist who’s been involved with the care of these patients for the balance of my career, it’s exciting to be a part of such cutting edge novel work for populations that mean so much to me. Cystic fibrosis is a progressive genetic disease. It affects the lung, but it also affects the pancreas and a myriad of other organs. There are close to 40,000 children and adults diagnosed and living with CF in the United States, and there’s estimated to be over a hundred thousand of them worldwide. In fact, this number is probably much larger. There was an analysis done in the early 2020s estimating about 160,000 people with CF living across over 90 countries. It’s estimated to be much higher because only about 65 percent of cases are appropriately diagnosed. It’s one of the most common genetic disorders among Caucasians. One in 30 people in the U.S. is a genetic carrier, and it occurs in one out of 3,200 live births. It’s also worth noting that it can affect people of every racial and ethnic group. It affects about one in 17,000 African Americans, one in 31,000 Asian Americans, and although universal newborn screening in the United States has helped us identify infants soon after birth, in many parts of the world, unfortunately, newborn screening is unavailable. So the diagnosis is oftentimes either missed or seriously delayed because of the lack of newborn screening available to other jurisdictions across the world, sadly.

Daniel Levine: And how does the condition manifest itself and progress?

Alan Cohen: So, CF affects a wide range of organ systems, as I mentioned, making people with the disease more likely to develop a variety of health conditions, including irreversible obstructive lung disease with a condition called bronchiectasis, which is an inflammatory process that chronically scars and widens and impacts lung function and allows for the overwhelming chronic lung infections that people with CF persist and live with throughout life. Insulin dependent diabetes, cirrhosis of the liver, arthritic conditions, infertility, and even osteoporosis are among many of their medical conditions that they suffer with. CF is the result of genetic mutations in what’s called the CFTR or cystic fibrosis transmembrane conductance regulator. It’s quite a mouthful. It’s on chromosome number seven and it’s what makes CFTR protein. There’s more than 2000 known genetic mutations within this CFTR genome, and as such, you can imagine there’s a wide spectrum of medical signs and symptoms associated with CF. Every healthy individual normally has two functional copies of the CFTR gene, and a person must have mutations in both copies to have the disease. Although the quality of life and survival of those suffering with CF has increased dramatically over the time of my career over the last 30 years, going from an average of living to not quite 20 years of age to well over 50 years of age today because of better and more directed medical treatment options. It’s worth noting that there’s currently no cure for people with CF and having been a former lung transplantation physician at Wash U in St. Louis, I personally was involved with lung transplantation as well as liver/lung transplantations for the majority of our CF patients who were able to make it to transplant. It still remains the only therapeutic option for those with more advanced disease today, obviously an imperfect cure, if at all.

Daniel Levine: We’ve seen great advances in therapeutic options for most people with the condition. You’re pursuing a gene therapy for patients who are ineligible for CFTR modulator therapy. One of the challenges with cystic fibrosis is the large number of mutations that can cause the condition, something on the order of 2000 different mutations. Is your gene therapy expected to work across mutations or be for a very specific one?

Alan Cohen: Yeah, that’s a great question and actually I think an important question. In fact, yes, 4D-710 should work across all subtypes. As you mentioned, there are certainly an enormous number of CFTR mutations, over 2000 within the CFTR gene. The commonality among all of these genetic mutations is a lack of sufficient and or functional CFTR protein and 4DMT’s approach was to create an investigational genetic medicine, which we refer to as 4D-710. It uses that A101 vector we’ve spoken about a moment ago for the treatment of lung disease. This is currently in phase 2 study for adults with CF who are ineligible for CFTR modulators or simply couldn’t tolerate them and discontinued their use because of intolerability or safety effects. It’s highly differentiated because it was designed for a single dose aerosol delivery containing a corrected transgene CFTR∆R, which we deliver directly to the airway. And although we’re currently targeting those people with CF lung disease ineligible or intolerant of current modulator therapies, there’s no reason to think that 4D-710 couldn’t be potentially beneficial to all genetic subtypes of people suffering with CF lung disease irrespective.

Daniel Levine: What’s known about it from studies you’ve done to date.

Alan Cohen: So, the clinical phase 1/2 study we’re currently running is called the AEROW study. It’s currently enrolling subjects in the United States, and we’re excited about the support that we’ve gotten from both the CF Foundation in the United States and their therapeutic research development network. Primary endpoints of the study are safety, tolerability, and defining a maximal tolerated dose as well as selection of a dose to expand into phase 2. Secondary endpoints as you would imagine, include assessments of clinical activity, quality of life and lung function, plus exploratory endpoints, assessing the feasibility of detecting transgene transfer and CFTR transgene expression, and we’re doing that via bronchoscopic biopsies and airway cell brushings. To date, we’ve treated nine people with CF across four different dosing levels with the goal of demonstrating safe delivery of our CFTR∆R genetic medicine. And at the National CF meeting, which was just a few months ago in the last quarter of 2023, our KPI presented biopsy data collected in the first seven of these subjects where we reported consistent high level expression of the CFTR∆R gene and CFTR protein across all samples collected, and the therapy by and large was generally well tolerated.

Daniel Levine: And what’s the development path forward?

Alan Cohen: So, we’re very excited about this and we’re thrilled to have a lot of interest throughout the United States and actually internationally. So, in this clinical phase 1/2, we currently have nine subjects enrolled in four different dosing groups, and regarding the first three subjects in cohort one, we’ve shared follow-up data on functional measures, and we observed stable or actually improved lung function as measured by spirometry looking at a measure called percent predicted FEV1. We’ve also shown improved quality of life as measured by a special quality of life measures specifically for people with CF called the CF Quality of Life questionnaire. Given these promising early findings, we’re working towards selecting a dose to expand into the phase 2 portion of the study to better understand this early signal. And if those trends continue and we hope they do, we will plan to enter into a pivotal study sometime in the second half of 2025 to get this important genetic medicine to people with cystic fibrosis as soon as possible. Also, with the high levels of gene delivery we’ve seen to date, we’re excited to continue building our pulmonary pipeline with our next generation program directed to the lung, 4D-725, which is targeting alpha 1 antitrypsin deficiency lung disease, another rare pulmonary disorder with high unmet medical needs despite good current standards of care.

Daniel Levine: You also have a program in Fabry disease that I wanted to ask you about. For listeners not familiar with Fabry, what is it?

Alan Cohen: Yeah, that’s a great question and you wouldn’t be surprised that they may not be familiar with Fabry’s disease. Just like cystic fibrosis, it’s quite a rare condition. It’s a monogenetic condition caused by mutations in the GLA gene, which is on the X chromosome. It encodes for alpha-galactosidase-A or AGA enzyme, and that results in the body’s inability to produce sufficient AGA enzyme, and that causes the accumulation of toxic levels of lipids, specifically glycolipids such as Lyso-Gb3, and this deposits in critical organs, sadly mostly in the heart and in the kidneys and blood vessels impacting the peripheral nervous system. Fabry disease, as I mentioned, is an orphan or rare disease and much like cystic fibrosis has a very low frequency, something like one in 40,000 men, something like one in over 100,000 females. 4D-310 was invented to deliver the GLA gene systematically across multiple organs, but unlike current therapies, we’re focusing on the heart disease, which other Fabry therapies had been unable to address. C102 is our next generation AAV that we have invented for targeting delivery to the cardiomyocytes for this genetically driven heart condition. And that’s seen in Fabry disease.

Daniel Levine: Like a lot of lysosomal storage disorders, Fabry’s been treated with enzyme replacement therapy. What are the limits of enzyme replacement therapies?

Alan Cohen: Yeah, and you make a very good point, and in fact, that’s one of the reasons why we’re also thinking about targeting alpha-1 antitrypsin deficiency, another disease which targets with replacement enzyme therapy, which you would think in theory should work, but in practice actually doesn’t make a very big difference in the organ systems that matter. The current standard of care in Fabry diseases [is] ERT or enzyme replacement therapy, or there’s an oral chaperone therapy that can restore or replace function of these lipids, but neither of them really address cardiomyopathy, which is the leading cause of death, and that accounts for about three quarters of the reason that people die and succumb to Fabry disease itself. That substrate accumulates in the heart muscle cells and that leads to life-threatening heart failure, dysrhythmias and vascular blockage. It’s a progressive and fatal disease with an average life expectancy sadly of only about 50 years. Progression of the disease causes significant reduction in quality of life, and as you can imagine, that has significant economic burden with greater patient needs for supportive care. Cardiac benefit has just simply not been demonstrated following the use of enzyme replacement therapy, which requires intravenous administration to the blood with either ERT or even more recently developed pegylated forms of ERT to elongate their half-life, or even gene therapies with enzyme expression targeting the liver. These remain meaningful limitations for current standard of care, and they simply don’t appear to meaningfully impact the leading cause of death in Fabry disease—that being cardiomyopathy and the heart related diseases due to substrate accumulation in the cardiomyocytes.

Daniel Levine: In constructing your therapy, was the key to be able to deliver it to cells in the heart?

Alan Cohen: Yeah, that’s exactly, so it’s an investigational genetic candidate. I mentioned using the C102 vector and in fact, that received Fast Track designation by the FDA in 2020 for the treatment of classic severe Fabry disease cardiomyopathy. In particular, it’s being evaluated in two phase 2 clinical trials that we’re calling INGLAXA targeting adults with classic or late-onset Fabry’s disease. We believe that this genetic medicine candidate is highly differentiated because it’s designed to be administered as a single low dose, intravenous genetic medicine to be delivered to and result in GLA or transgene expression within the actual heart muscle cells itself. It directly targets cardiomyocytes, and that’s what’s unique about it, and that is intended to specifically correct this leading cause of mortality in the heart muscle itself.

Daniel Levine: And what’s known about it from studies to date and what’s the development path forward?

Alan Cohen: Yeah, great question. To date, we’ve treated six subjects with Fabry’s disease and we’ve shown promising trends across multiple approvable cardiac endpoints for heart function and quality of life, and that’s simply not a bar that anyone has hit thus far. We’ve also examined cardiac biopsies from a single patient confirming that there was gene delivery to the cardiomyocytes in this treated subject. In terms of next steps, we’re currently on clinical hold due to a safety event called aHUS, or atypical hemolytic uremic syndrome, and that was due to administration of the therapy. And of note, this is a known class effect for IVA AAV therapies. The therapy was well tolerated after this initial aHUS period. However, we’re testing an alternative immune-prophylactic regimen using rituximab and sirolimus, two immunosuppressive drugs. I have a lot of experience within the transplant world, and these have been successfully used to prevent aHUS establishment in other subjects and other AAV studies. Historically, we’ll be submitting our preclinical data regarding this immunosuppressive regimen to the FDA in the coming quarter, and then we hope to be coming off of clinical hold shortly thereafter. We also look forward to working with the FDA to find a path forward and to get this genetic medicine to Fabry patients as quickly as possible.

Daniel Levine: Given your unique collection of vectors and your ability to generate them and the reality of the limits of any single company to develop a pipeline of gene therapies, it would seem that licensing and partnering deals would be an important component of your strategy. How does this fit into 4DMT’s business model?

Alan Cohen: Yeah, that’s a great question and now I’ll put on my business hat. Licensing and partnering has been a key part of our business in the past and will continue to be in the future. However, strategically as a company, we’re in a very strong position, unlike many where we have the financial strength and capabilities to control our own destiny. In fact, that’s why I joined the company. The challenge of us moving forward is focus and prioritization to ensure the success of our highest potential development programs and to consider strategic partnerships for areas where we have less capabilities to ensure those genetic medicines are in the best hands to get to patients the soonest and to maximize their value. In addition to product portfolio, our vectors themselves have tremendous value, so we’re very open to doing licensing deals that require minimal internal efforts, but provide our partners the ability to access and employ them to deliver their own genetic medicines in exchange for cash up front and milestones, for example, which is great for the entire genetic medicine field since it allows our technologies’ potential to be maximized. Our deal with R100, for example, with Astellas in rare eye diseases, is a perfect example of an ideal partnership of that nature.

Daniel Levine: You mentioned the value of your vectors. This is always a challenge in a partnership when you’re contributing something like a vector, is to get agreement and recognition of the value. Has this been an issue in forging these deals?

Alan Cohen: Increasingly, the value of next generation targeted and customizable AAV vectors is being recognized. The R100 transaction, as I mentioned a moment ago, with Astellas was, to our knowledge, one of the highest value deals for a structure of that type with access to our technology rather than an entire product candidate that has already been through preclinical studies. For example, more recently, our deal with Arbor was a 50/50 economic collaboration where we provide the best capsid, they provide the best gene editing payloads, and once again, it highlights the relative value of the delivery vehicle versus the payload in a genetic medicine product. Both of these business models are great ways to maximize the utilization of complementary technologies to push the field forward and ultimately, in my perspective, to benefit patients with the best possible genetic medicines. And that’s what really 4D is all about. And as I mentioned earlier, that’s why I joined their ranks and I’m thrilled to be here.

Daniel Levine: Alan Cohn, senior vice president of Clinical Development and Therapeutic Area, head of pulmonology for 4DMT. Alan, thanks so much for your time today,

Alan Cohen: Danny. Thank you so much. Pleasure talking to you.

This transcript has been edited for clarity and readability.


The RARECast podcast is made possible through support from the Global Genes’ Corporate Alliance. The members of the Corporate Alliance support Global Genes’ mission and programs, work to meet the vital needs of people with rare diseases, and address inequities they face. To learn more about the Corporate Alliance or how your organization can become a member, click here.


Stay Connected

Sign up for updates straight to your inbox.