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Cell & Gene Therapy Insights

Cell & Gene Therapy Insights

Issue Vol 5 Suppl. 1


DNA Vectors – a Key Enabler of Tomorrow’s Cancer Immunotherapies


Richard Paul Harbottle


Richard Paul Harbottle is currently the Principle Investigator and Group Leader of the DNA Vector Research Group at the German Cancer Research Centre (DKFZ) in Heidelberg. Their research focuses on the development of DNA vector technologies for gene therapy with particular focus on the application of minimally sized scaffold/matrix attachment region (S/MAR) vectors. They have recently made a breakthrough in their DNA vector design that allows the application of our vector in stem cells and in primary human cells. For the first time they have generated a DNA vector system that can provide persistent transgene expression in primary human T cells without the risk of integration-mediated genotoxicity and they are currently developing a range of novel DNA vectors for anti-tumor immunotherapy. This DNA vector can also be used to genetically modify stem cells and they have recently shown for the first time that they can generate stable mouse embryonic cell lines and can generate transgenic mice from these modified cells.

Richard, can you give us a brief background on your career to date in the non-viral gene therapy area?

I started my efforts in non-viral gene therapy in the early ‘90s, when I started a Master’s degree at St Mary’s Hospital Medical School, now part of Imperial College. I went to do my Master’s degree in human molecular genetics in the department of Professor Bob Williamson. He was effectively a gene hunter; they were looking for the genes that cause disease. In particular, at the time he was looking for thegene for cystic fibrosis (CF) and they got close to finding it, and certainlyhad it cloned in the fridge, but it was characterized by another group in the late 1980s.

When I arrived in the beginning of the 1990s to St Mary’s, they’d already started talking about gene therapy for CF and the field was very young then, we weren’t really sure what to do, but there were big interests in developing non-viral gene therapy for CF.

The CF research continued way through the ‘80s and ‘90s, and the CF Consortium in the UK concentrated on non-viral gene therapy for a long time, all the way through to clinical trials. So my Master’s degree and my PhD was involved in trying to use non-viral constructs to get DNA into cells. And particularly into the airway epithelia.

It was really rather difficult in those times. We didn’t understand how we could get DNA into cells, certainly in vivo.

Then through the ‘90s we actually managed to become much more successful at delivering DNA in vivo. Then we realized although the delivery was improving, the DNA vectors were not. So the non-viral constructs we were using were not as effective as they needed to be.

So after my time trying to deliver DNA into cells, then I started work on trying to improve the DNA itself to make it more efficient for gene therapy.And to bring us up to date, what are you working on now in your lab?

We’re still working on non-viral constructs, so really we’re a non-viral vector lab focused on developing DNA that is effective in a range of applications for gene and cell therapy.

One crucial change in our approach was when we came to realize that not being a virus was not enough to enable effective gene delivery, or gene expression. We always used to think non-viral vectors would be more effective because they were loss toxic and less damaging to the cells. In fact, we thought the DNA was inert and should not be pathogenic or damaging in any way. It turns out that’s not really true.

I think one of the big obstacles for gene therapy is the delivery; human and mammalian cells are designed to protect themselves from infection. And we always thought viruses could cause damage to cells by being an infectious particle, by being a human pathogen. There would be immunological consequences, inflammatory consequences, because it’s a virus. And we didn’t really think or consider at the time that DNA, a circle of DNA, whatever format the DNA is being transfected into the cell could also antagonize the cell.

It turns out that there are not only barriers on the outside of the cell, but lots of sensors and other mechanisms a cell has to defend itself from bacterial and viral infection. And essentially the transfection or nucloporation, or however you deliver some DNA to the cell, the DNA has to come from the outside of the cell, translocate through the cytoplasm and into the nucleus. And that in itself can be detected by the cell as an infective process. The cell has many sensors in the cytoplasm and the nucleus to detect DNA in the wrong place and it can defend itself by inflammatory reactions.

What we’ve been working on is trying to get an improved DNA vector that does not antagonize the cell, that does not cause any molecular damage, to better understand the consequences of transfection and gene delivery to a cell, and to try and overcome that.

The vectors our lab is working on right now overcome many of these limitations. We can genetically modify a cell persistently, without causing molecular or genetic damage. Our DNA exists episomaly in a cell, and does not antagonize the innate immunity of the cell.

As we’ve overcome some of these issues we’re able to apply it to a range of cell models and in vivo models, which we were not capable of attempting before. We’re working on developing this vector system and applying it in a range of new approaches for ex vivo cell therapy as well as gene therapy in vivo.

On the topic of applications, can you tell us about the potential non-viral vectors hold specifically for the cellular immunotherapy space?

I think the immunotherapy field is very exciting at the moment.

As you know there are cellular drugs that have been approved, and the genetic modification of human T cells to target them against tumors is proving to be effective and is a very exciting field to be in.

Typically this is done using a lentiviral vector, which can introduce a CAR, a recombinant T-cell receptor, into T cells, and target it against CD19 leukemia’s.

I think there’s plenty of scope if you can develop an alternative vector system that could be used in this space. The difficulty is in achieving sufficient expression in the cells. Notdamaging the T cells so they can perform the duty, target tumors when they’re reintroduced into the patient. And I think there’s certainly great advances in non-viral applications for human immunotherapy.

There’s great work in the USA and in Europe to develop sleeping beauty vectors that can be used for CAR-T immunotherapy. We’re following by using our vector system, which does not integrate, it requires one vector only for CAR-T immunotherapy. And for these drugable, targetable tumors, I think CAR-T immunotherapy is going to be a very exciting field in the coming years.

What will be the chief challenges or key milestones to achieve before that potential can be realized?

The field is increasingly advanced. I think the protocols for introducing CAR-Ts using viruses is well established; however, the kinetics of the process are different for non-viral constructs. If you were using a sleeping beauty construct then the T cells need to be in a different condition and need slightly different culturing procedures.

One of the big obstacles is the introduction of DNA into these T cells. And there are several companies now that manufacture devices that can do clinical-grade and -scale nucleofection of T cells. We’re working with some of these companies to try and improve the introduction of the DNA. I think the DNA is at this stage whereby the vectors themselves are capable of genetically modifying T cells to a state where they can be used for CAR-T immunotherapy.

The large clinical-scale introduction of DNA into these cells is one of the biggest barriers now and I think the developments in the technology for maintaining T cells are starting to address this. Miltenyi is one example, their Prodigy machine can essentially look after the T cells, expand the T cells, purify the T cells, and keep them in a clinical GMP quality space for reintroduction into the patient. And they have a very sophisticated electroporation device which is compatible with this, which can do large- scale nucleoporation.

We’re also working with other companies like Lonza and MaxCyte to try and introduce our DNA into cells. And both of them have large, clinical-scale electroporation devices.

Another critical issue is that the cell biology needs to be understood a bit better. There’s still a division of opinion whether T cells need to be persistently modified. Whether the T cells should go into a memory stateand survive after the initial immunotherapy treatment. And with using these integrating vectors, you could then potentially develop a T- cell population that remembers the CART, and will then monitor tumours after the initial administration.

So we’re not sure whether we need to have T cells that are persistently modified forever, and if they are persistently modified then there’s the issue of safety. Is the integration event safe, long term? And if we’re not integrating, do we really need persistence? Do we need lifetime genetic modification of these T cells?

The challenge is to understand better how long we need to modify these T cells, the safest way to do it. But certainly for non-viral vectors, the biggest challenge we have at the moment, because we’re quite convinced the DNA vectors work, is the large-scale clinical grade transfection of them.

Non-viral delivery seems to be enjoying something of a resurgence overall. What for you are essential to ensuring the field continues to progress and translates into effective therapeutic modalities?

This really goes back to the early work. If we’re talking about gene therapy, so the in vivo, or introduction of genetic vectors into a patient, they’re going to require some sophisticated gene delivery strategies.

And although there are many approaches now with nanoparticles and other formulations to effectively deliver DNA into a tissue or specifically into a tumor, then these are still not as effective as an infective viral particle. So there needs to be continued development on the gene delivery side. And in fact many approaches at the moment involve actually the ex vivo modification of cells.

So we’re, even with viruses, taking a cell out of a patient, genetically modifying, and then reintroducing the cell, is typically common in both viral and non-viral field. So we’ve caught up certainly for the ex vivo approach because that’s much more simple, genetically modifying a cell in a culture, or in a dish rather than in a patient.

So I think the gene delivery protocols have to be improved. Our formulations have to be more effective, more efficient, and I think there’s still lots of work to be done with that.

I think the vectors themselves it’s clear that there’s plenty of scope. And one of the reasons there is more interest, or continued interest in non-viral approaches, is their flexibility. Certainly the viral approaches for gene therapy, they go through waves. We’ve had adenovirus, integrating lenti and
retroviruses, and now really we’re using AAV.

But there’s always a limitation by using some of these viral particles, in particular the immunological issues with AAV and also their capacity. So we are limited by the administrative routes we can use and the genes we can deliver with AAV.

With a non-viral approach there is no limitation on capacity so we’ve got much more scope for designing more sophisticated genetic packages. And we can deliver large genes, we can deliver better designed constructs, we’ve got regulatory sequences, we’ve got sequences which provide endogenous expression and even safety switches. There’s lots of scope in non-viral vector design where we can make more sophisticated genetics.

In addition the production of these is much more simple and cheaper. We can make huge amounts of DNA at very high quality, even GMP quality. And in fact for the CAR-T immunotherapy, in principle we could make grams of DNA, which would satisfy the clinical requirements for several years with one single batch of DNA, for a drugable construct.

Say for example we’re going to make a CD19 CAR-T vector, we could make enough to be used in every single clinical trial for the coming years from one single batch. And the batch of DNA is much more easy to store. We can freeze it, we can lyophilise it, and we’ve got really high purity. So simple production. Ease of preparation and ease of storing. And the large-scale quantities we can make non-viral vectors, or certainly the DNA component of a non-viral vector. I think that is certainly going to be something for the future, and something that has kept non-viral vectors as a viable alternative to the viruses in the gene and cell therapy space.


Richard Paul Harbottle
Head, Research Group DNA Vectors, German Cancer Research Centre
Heidelberg, Germany

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In vivo electroporation: the key to unlocking the power of DNA-encoded vaccines and monoclonal antibodies


Trevor Smith


Trevor Smith is a molecular and cellular immunologist, whose research focuses upon the delivery of drugs against infectious disease and oncology targets. He is the lead or co-author of over 30 research papers across multiple fields. Dr Smith heads a preclinical research team developing DNA-based prophylactics and therapeutics at Inovio Pharmaceuticals in San Diego, California. He received his Bachelor degree in Biochemistry and Immunology from King’s College London, and completed his PhD studies at the National Heart and Lung Institute, Imperial College London. Dr Smith was the recipient of postdoctoral fellowships at the Scripps Research Institute in La Jolla, California.

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Progressing Non-Viral Gene Therapy Approaches in GI Tract & Bladder Diseases


Anthony Cheung


Anthony Cheung is the co-founder and Chief Technology Officer of enGene, a private biotechnology company headquartered in Montreal, Canada. He served as the Chief Executive Officer of enGene from 2012 to 2018. Dr Cheung is a co-inventor of enGene’s non-viral gene delivery platform for mucosal tissues and its Gene PillTM technology. During his tenure as the CEO, he raised significant financing to grow the company through venture-backed equity financing and government funding. He also successfully completed two partnership transactions with Johnson & Johnson and Takeda. enGene was awarded by BIOTECanada in 2017 as the Biotech Company of the Year. Dr Cheung was recognized in 2016 by Biotechnology Focus, a Canadian life science-focused trade publication, as one of the Top 5 Biotech CEO in Canada and selected as the EY Entrepreneur of the Year Finalist in 2017. Dr Cheung received his doctorate degree in Physiology from the Tulane University School of Medicine in New Orleans. Dr Cheung has co-authored numerous book chapters, review articles and peer-reviewed journals on the topics of diabetes, gene therapy and autoimmune diseases. His research has been published in many prestigious scientific journals, including Science and Proceedings of the National Academy of Science. He has been invited to speak at many international scientific and biotechnology conferences – BIO, American Society for Gene & Cell Therapy, Diabetes Technology Meeting, Children with Diabetes – on topics related to gene therapy, diabetes and bio-entrepreneurism. He also serves as Board Member and Advisor for several biotechnology companies and professional organizations including Bio-Industry Liaison Committee of the American Society for Gene and Cell Therapy and Student Biotechnology Network of BC.

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The Future of Non-Viral Delivery: Niche Player or Gene Therapy Cornerstone?



Lynn Zechiedrich


Lynn Zechiedrich is the Kyle and Josephine Morrow Chair and Professor in Molecular Virology and Microbiology at Baylor College of Medicine. She developed minivectors to study DNA, the enzymes that act on DNA, and the antibiotic and anticancer drugs that inhibit these enzymes. Minivectors also proved to be excellent gene therapy delivery vectors. Among other honors, she won a New Investigator Award from the Burroughs Wellcome Fund, a Curtis Hankamer Research Award, and funding from the Human Frontier Science Program. She is a Fellow of the National Academy of Inventors. She was Baylor College of Medicine’s BRASS Mentor of the Year in 2013. She holds two issued US patents and three issued foreign patents that are licensed to Twister Biotech, Inc., a company she founded in 2011, and has multiple patents pending. She has published more than 60 articles and book chapters and given over 170 invited talks. She served on numerous grant review committees, reviews for 40 different peer-reviewed journals, ranging from mathematics and physics to microbiology and gene therapy, and serves on multiple editorial boards.

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Innovator Insight

Non-Viral Vector Mediated Gene Delivery: the Outsider to Watch Out For in Gene Therapy

Innovator Insight

Alengo Nyamay’Antu, Maxime Dumont, Valérie Kedinger & Patrick Erbacher


Nucleic acids have considerable potential as therapeutic agents in the treatment of pathologies including genetic diseases, viral infections and cancer therapies. The major challenge for the use of nucleic acids in therapy lies in safely delivering these anionic macromolecules to their intended sites of action. The increasing use of viral vectors in Human Gene Therapy clinical trials has emphasized the potential of nucleic acid-based approaches to address the unmet needs of drug-based treatments. While viral vector-based Gene Therapy is on everyone’s mind with recently approved Luxturna™, as well as other viral vector-based treatments under Fast Track Designation, it is key to remember that non-viral vectors present considerable advantages in terms of reliability, safety and costs for nucleic-acid based therapies. At Polyplus-transfection®, we develop powerful non-viral vectors to safely deliver nucleic acids in vivo to target a wide range of tissues, through various routes of administrations. Of these reagents, in vivo-jetPEI® and its highest quality grade cGMP in vivo-jetPEI® are acknowledged as a non-viral vector of choice to deliver nucleic acids respectively in animal preclinical studies and in human clinical trials, notably for cancer and immunotherapy.

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