For more in-depth learning, we recommend Different Approaches in our Patient Education program.

The challenges of gene and cell therapists can be divided into three broad categories based on disease, development of therapy, and funding.

Challenges based on the disease characteristics: Disease symptoms of most genetic diseases, such as Fabrys, hemophilia, cystic fibrosis, muscular dystrophy, Huntingtons, and lysosomal storage diseases are caused by distinct mutations in single genes. Other diseases with a hereditary predisposition, such as Parkinsons disease, Alzheimers disease, cancer, and dystonia may be caused by variations/mutations in several different genes combined with environmental causes. Note that there are many susceptible genes and additional mutations yet to be discovered. Gene replacement therapy for single gene defects is the most conceptually straightforward. However, even then the gene therapy agent may not equally reduce symptoms in patients with the same disease caused by different mutations, and even the samemutationcan be associated with different degrees of disease severity. Gene therapists often screen their patients to determine the type of mutation causing the disease before enrollment into a clinical trial.

The mutated gene may cause symptoms in more than one cell type. Cystic fibrosis, for example, affects lung cells and the digestive tract, so the gene therapy agent may need to replace the defective gene or compensate for its consequences in more than one tissue for maximum benefit. Alternatively, cell therapy can utilizestem cellswith the potential to mature into the multiple cell types to replace defective cells in different tissues.

In diseases like muscular dystrophy, for example, the high number of cells in muscles throughout the body that need to be corrected in order to substantially improve the symptoms makes delivery of genes and cells a challenging problem.

Some diseases, like cancer, are caused by mutations in multiple genes. Although different types of cancers have some common mutations, every tumor from a single type of cancer does not contain the same mutations. This phenomenon complicates the choice of a single gene therapy tactic and has led to the use of combination therapies and cell elimination strategies. For more information on gene and cell therapy strategies to treat cancer, please refer to the Cancer and Immunotherapy summary in the Disease Treatment section.

Disease models in animals do not completely mimic the human diseases and viralvectorsmay infect various species differently. The testing of vectors in animal models often resemble the responses obtained in humans, but the larger size of humans in comparison to rodents presents additional challenges in the efficiency of delivery and penetration of tissue.Gene therapy, cell therapy, and oligonucleotide-based therapy agents are often tested in larger animal models, including rabbit, dog, pig and nonhuman primate models. Testing human cell therapy in animal models is complicated by immune rejections. Furthermore, humans are a very heterogeneous population. Their immune responses to the vectors, altered cells, or cell therapy products may differ or be similar to results obtained in animal models.

Challenges in the development of gene and cell therapy agents: Scientific challenges include the development of gene therapy agents that express the gene in the relevant tissue at the appropriate level for the desired duration of time. There are a lot of issues in that once sentence, and while these issues are easy to state, each one requires extensive research to identify the best means of delivery, how to control sufficient levels or numbers of cells, and factors that influence duration of gene expression or cell survival. After the delivery modalities are determined, identification and engineering of a promoter and control elements (on/off switch and dimmer switch) that will produce the appropriate amount of protein in the target cell can be combined with the relevant gene. This gene cassette is engineered into a vector or introduced into thegenomeof a cell and the properties of the delivery vehicle are tested in different types of cells in tissue culture. Sometimes things go as planned and then studies can be moved onto examination in animal models. In most cases, the gene/cell therapy agent may need to be improved further by adding new control elements to obtain the desired responses in cells and animal models.

Furthermore, the response of the immune system needs to be considered based on the type of gene or cell therapy being undertaken. For example, in gene or cell therapy for cancer, one aim is to selectively boost the existing immune response to cancer cells. In contrast, to treat genetic diseases like hemophilia and cystic fibrosis the goal is for the therapeutic protein to be accepted as an addition to the patients immune system.

If the new gene is inserted into the patients cellularDNA, the intrinsic sequences surrounding the new gene can affect its expression and vice versa. Scientists are now examining short DNA segments that may insulate the new gene from surrounding control elements. Theoretically, these insulator sequences would also reduce the effect of vector control signals in the gene cassette on adjacent cellular genes. Studies are also focusing on means to target insertion of the new gene into safe areas of the genome, to avoid influence on surrounding genes and to reduce the risk of insertional mutagenesis.

Challenges of cell therapy include the harvesting of the appropriate cell populations and expansion or isolation of sufficient cells for one or multiple patients. Cell harvesting may require specific media to maintain the stem cells ability toself-renew and mature into the appropriate cells. Ideally extra cells are taken from the individual receiving therapy. Those additional cells can expand in culture and can be induced to becomepluripotent stem cells(iPS), thus allowing them to assume a wide variety of cell types and avoiding immune rejection by the patient. The long term benefit of stem cell administration requires that the cells be introduced into the correct target tissue and become established functioning cells within the tissue. Several approaches are being investigated to increase the number of stem cells that become established in the relevant tissue.

Another challenge is developing methods that allow manipulation of the stem cells outside the body while maintaining the ability of those cells to produce more cells that mature into the desired specialized cell type. They need to provide the correct number of specialized cells and maintain their normal control of growth and cell division, otherwise there is the risk that these new cells may grow into tumors.

Challenges in funding: In most fields, funding for basic or applied research for gene and cell therapy is available through the National Institutes of Health (NIH) and private foundations. These are usually sufficient to cover the preclinical studies that suggest a potential benefit from a particular gene and cell therapy. Moving into clinical trials remains a huge challenge as it requires additional funding for manufacturing of clinical grade reagents, formal toxicology studies in animals, preparation of extensive regulatory documents, and costs of clinical trials.Biotechnology companies and the NIH are trying to meet the demand for this large expenditure, but many promising therapies are slowed down by lack of funding for this critical next phase.

Read more:

Gene & Cell Therapy FAQs | ASGCT - American Society of Gene & Cell ...

An Air Force medical technician draws a dose of the COVID-19 vaccine to inoculate Air Force reservists at Joint Base Lewis McChord, Washington, Sept. 12, 2021. (U.S. Air Force photo by Staff Sgt. Paolo Felicitas)

Many skeptics have contended that the mRNA-based Pfizer and Moderna COVID-19 shots are not "vaccines" but rather a form of gene therapy that poses untold risks by altering a recipient's DNA.

The federal government and health-care experts have denied that claim. But the president of Bayer's Pharmaceuticals Division is on record describing the mRNA shots as "cell and gene therapy" and acknowledging public wariness of the technology.

Bayer executive Stefan Oelrich, LifeSiteNews reported, made the statement at the World Health Summit, which took place in Berlin Oct. 24-26, drawing 6,000 people from 120 countries.

Oelrich said his company is "really taking that leap" to drive innovation "in cell and gene therapies."

"Ultimately, the mRNA vaccines are an example for that cell and gene therapy," he said.

"I always like to say: If we had surveyed two years ago in the public 'would you be willing to take a gene or cell therapy and inject it into your body?' we probably would have had a 95% refusal rate," Oelrich said.

In August, Reuters ran a "fact check" citing experts who contend that the technology in the Pfizer/BioNTech and Moderna shots are not gene therapy.

Both shots usea piece of genetic code from SARS-CoV-2 to prompt an immune response in recipients. But Dr. Adam Taylor, a virologist and researcher at Griffith University in Australia, insisted that while it's "a genetic-based therapy," it doesn't alter a person's genes.

Gene therapy, in the classical sense, involves making deliberate changes to a patients DNA in order to treat or cure them," he said. "mRNA vaccines will not enter a cells nucleus that houses your DNA genome. There is zero risk of these vaccines integrating into our own genome or altering our genetic makeup."

At the Berlin summit, the Bayer executive said that his company's "successes" over the 18 months of the pandemic "should embolden us to fully focus much more closely on access, innovation and collaboration to unleash health for all, especially as we enter, on top of everything else that is happening, a new era of science a lot of people talk about the Bio Revolution in this context."

LifeSiteNews noted that, according to the McKinsey Global Institute, the "Bio Revolution" is "a confluence of advances in biological science and accelerating development of computing, automation, and artificial intelligence [that] is fueling a new wave of innovation."

"This Bio Revolution could have significant impact on economies and our lives, from health and agriculture to consumer goods, and energy and materials."

Oelrich said Bayer also is working at reducing the populations of Third World countries, investing $400 million in "long-acting contraceptives" and partnering with the Bill and Melinda Gates Foundation on "family planning initiatives."

EDITOR'S NOTE: Last year, America's doctors, nurses and paramedics were celebrated as frontline heroes battling a fearsome new pandemic. Today, under Joe Biden, tens of thousands of these same heroes are denounced as rebels, conspiracy theorists, extremists and potential terrorists. Along with massive numbers of police, firemen, Border Patrol agents, Navy SEALs, pilots, air-traffic controllers, and countless other truly essential Americans, they're all considered so dangerous as to merit termination, their professional and personal lives turned upside down due to their decision not to be injected with the experimental COVID vaccines. Bidens tyrannical mandate threatens to cripple American society from law enforcement to airlines to commercial supply chains to hospitals. It's already happening. But the good news is that huge numbers of "yesterdays heroes" are now fighting back bravely and boldly. The whole epic showdown is laid out as never before in the sensational October issue of WND's monthly Whistleblower magazine, titled "THE GREAT AMERICAN REBELLION: 'We will not comply!' COVID-19 power grab ignites bold new era of national defiance."

Content created by the WND News Center is available for re-publication without charge to any eligible news publisher that can provide a large audience. For licensing opportunities of our original content, please contact licensing@wndnewscenter.org.

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Overview

Gene therapy involves altering the genes inside your body's cells in an effort to treat or stop disease.

Genes contain your DNA the code that controls much of your body's form and function, from making you grow taller to regulating your body systems. Genes that don't work properly can cause disease.

Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve your body's ability to fight disease. Gene therapy holds promise for treating a wide range of diseases, such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS.

Researchers are still studying how and when to use gene therapy. Currently, in the United States, gene therapy is available only as part of a clinical trial.

Gene therapy is used to correct defective genes in order to cure a disease or help your body better fight disease.

Researchers are investigating several ways to do this, including:

Gene therapy has some potential risks. A gene can't easily be inserted directly into your cells. Rather, it usually has to be delivered using a carrier, called a vector.

The most common gene therapy vectors are viruses because they can recognize certain cells and carry genetic material into the cells' genes. Researchers remove the original disease-causing genes from the viruses, replacing them with the genes needed to stop disease.

This technique presents the following risks:

The gene therapy clinical trials underway in the U.S. are closely monitored by the Food and Drug Administration and the National Institutes of Health to ensure that patient safety issues are a top priority during research.

Currently, the only way for you to receive gene therapy is to participate in a clinical trial. Clinical trials are research studies that help doctors determine whether a gene therapy approach is safe for people. They also help doctors understand the effects of gene therapy on the body.

Your specific procedure will depend on the disease you have and the type of gene therapy being used.

For example, in one type of gene therapy:

Viruses aren't the only vectors that can be used to carry altered genes into your body's cells. Other vectors being studied in clinical trials include:

The possibilities of gene therapy hold much promise. Clinical trials of gene therapy in people have shown some success in treating certain diseases, such as:

But several significant barriers stand in the way of gene therapy becoming a reliable form of treatment, including:

Gene therapy continues to be a very important and active area of research aimed at developing new, effective treatments for a variety of diseases.

Explore Mayo Clinic studies of tests and procedures to help prevent, detect, treat or manage conditions.

Dec. 29, 2017

Excerpt from:

Gene therapy - Mayo Clinic

For Immediate Release: December 18, 2017

Espaol

The U.S. Food and Drug Administration today approved Luxturna (voretigene neparvovec-rzyl), a new gene therapy, to treat children and adult patients with an inherited form of vision loss that may result in blindness. Luxturna is the first directly administered gene therapy approved in the U.S. that targets a disease caused by mutations in a specific gene.

Todays approval marks another first in the field of gene therapy both in how the therapy works and in expanding the use of gene therapy beyond the treatment of cancer to the treatment of vision loss and this milestone reinforces the potential of this breakthrough approach in treating a wide-range of challenging diseases. The culmination of decades of research has resulted in three gene therapy approvals this year for patients with serious and rare diseases. I believe gene therapy will become a mainstay in treating, and maybe curing, many of our most devastating and intractable illnesses, said FDA Commissioner Scott Gottlieb, M.D. Were at a turning point when it comes to this novel form of therapy and at the FDA, were focused on establishing the right policy framework to capitalize on this scientific opening. Next year, well begin issuing a suite of disease-specific guidance documents on the development of specific gene therapy products to lay out modern and more efficient parameters including new clinical measures for the evaluation and review of gene therapy for different high-priority diseases where the platform is being targeted.Luxturna is approved for the treatment of patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy that leads to vision loss and may cause complete blindness in certain patients.

Hereditary retinal dystrophies are a broad group of genetic retinal disorders that are associated with progressive visual dysfunction and are caused by mutations in any one of more than 220 different genes. Biallelic RPE65 mutation-associated retinal dystrophy affects approximately 1,000 to 2,000 patients in the U.S. Biallelic mutation carriers have a mutation (not necessarily the same mutation) in both copies of a particular gene (a paternal and a maternal mutation). The RPE65 gene provides instructions for making an enzyme (a protein that facilitates chemical reactions) that is essential for normal vision. Mutations in the RPE65 gene lead to reduced or absent levels of RPE65 activity, blocking the visual cycle and resulting in impaired vision. Individuals with biallelic RPE65 mutation-associated retinal dystrophy experience progressive deterioration of vision over time. This loss of vision, often during childhood or adolescence, ultimately progresses to complete blindness.

Luxturna works by delivering a normal copy of the RPE65 gene directly to retinal cells. These retinal cells then produce the normal protein that converts light to an electrical signal in the retina to restore patients vision loss. Luxturna uses a naturally occurring adeno-associated virus, which has been modified using recombinant DNA techniques, as a vehicle to deliver the normal human RPE65 gene to the retinal cells to restore vision.

The approval of Luxturna further opens the door to the potential of gene therapies, said Peter Marks, M.D., Ph.D., director of the FDAs Center for Biologics Evaluation and Research (CBER). Patients with biallelic RPE65 mutation-associated retinal dystrophy now have a chance for improved vision, where little hope previously existed.

Luxturna should be given only to patients who have viable retinal cells as determined by the treating physician(s). Treatment with Luxturna must be done separately in each eye on separate days, with at least six days between surgical procedures. It is administered via subretinal injection by a surgeon experienced in performing intraocular surgery. Patients should be treated with a short course of oral prednisone to limit the potential immune reaction to Luxturna.

The safety and efficacy of Luxturna were established in a clinical development program with a total of 41 patients between the ages of 4 and 44 years. All participants had confirmed biallelic RPE65 mutations. The primary evidence of efficacy of Luxturna was based on a Phase 3 study with 31 participants by measuring the change from baseline to one year in a subjects ability to navigate an obstacle course at various light levels. The group of patients that received Luxturna demonstrated significant improvements in their ability to complete the obstacle course at low light levels as compared to the control group.

The most common adverse reactions from treatment with Luxturna included eye redness (conjunctival hyperemia), cataract, increased intraocular pressure and retinal tear.

The FDA granted this application Priority Review and Breakthrough Therapy designations. Luxturna also received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases.

The sponsor is receiving a Rare Pediatric Disease Priority Review Voucher under a program intended to encourage development of new drugs and biologics for the prevention and treatment of rare pediatric diseases. A voucher can be redeemed by a sponsor at a later date to receive Priority Review of a subsequent marketing application for a different product. This is the 13th rare pediatric disease priority review voucher issued by the FDA since the program began.

To further evaluate the long-term safety, the manufacturer plans to conduct a post-marketing observational study involving patients treated with Luxturna.

The FDA granted approval of Luxturna to Spark Therapeutics Inc. The FDA, an agency within the U.S. Department of Health and Human Services, protects the public health by assuring the safety, effectiveness, and security of human and veterinary drugs, vaccines, and other biological products for human use, and medical devices. The agency also is responsible for the safety and security of our nations food supply, cosmetics, dietary supplements, products that give off electronic radiation, and for regulating tobacco products.

Luxturna is the first gene therapy approved in the U.S. to target a disease caused by mutations in a specific gene

Andrea Fischer301-796-0393

888-INFO-FDAOCOD@fda.hhs.gov

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FDA approves novel gene therapy to treat patients with a rare form of ...

BioDrugs. 2017; 31(4): 317334.

1Janssen Research and Development, 200 McKean Road, Spring House, PA 19477 USA

1Janssen Research and Development, 200 McKean Road, Spring House, PA 19477 USA

1Janssen Research and Development, 200 McKean Road, Spring House, PA 19477 USA

2BiStro Biotech Consulting, LLC, Bridgewater, NJ 08807 USA

1Janssen Research and Development, 200 McKean Road, Spring House, PA 19477 USA

2BiStro Biotech Consulting, LLC, Bridgewater, NJ 08807 USA

Open AccessThis article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies. The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy.

The discovery of DNA as the biomolecule of genetic inheritance and disease opened up the prospect of therapies in which mutant, damaged genes could be altered for the improvement of the human condition. The recent ability to rapidly and affordably perform human genetics on hundreds of thousands of people, and to sequence complete genomes, has resulted in an explosion of nucleic acid sequence information and has allowed us to identify the gene, or genes, that might be driving a particular disease state. If the mutant gene(s) could be fixed, or if the expression of overactive/underactive genes could be normalized, the disease could be treated at the molecular level, and, in best case scenarios, potentially be cured. This concept seems particularly true for the treatment of monogenic diseases, i.e. those diseases caused by mutations in a single gene. This seemingly simple premise has been the goal of gene therapy for over 40years.

Until relatively recently, that simple goal was very elusive as technologies to safely deliver nucleic acid cargo inside cells have lagged behind those used to identify disease-associated genes. One of the earliest approaches investigated was the use of viruses, naturally occurring biological agents that have evolved to do one thing, i.e. deliver their nucleic acid (DNA or RNA) into a host cell for replication. There are numerous viral agents that could be selected for this purpose, each with some unique attributes that would make them more or less suitable for the task, depending on the desired profile [1]. However, the undesired properties of some viral vectors, including their immunogenic profiles or their propensity to cause cancer have resulted in serious clinical adverse events and, until recently, limited their current use in the clinic to certain applications, for example, vaccines and oncolytic strategies [2]. More artificial delivery technologies, such as nanoparticles, i.e. chemical formulations meant to encapsulate the nucleic acid, protect it from degradation, and get through the cell membrane, have also achieved some levels of preclinical and clinical success. Not surprisingly, they also have encountered some unwanted safety signals that need to be better understood and controlled [3].

Adeno-associated virus (AAV) is one of the most actively investigated gene therapy vehicles. It was initially discovered as a contaminant of adenovirus preparations [4, 5], hence its name. Simply put, AAV is a protein shell surrounding and protecting a small, single-stranded DNA genome of approximately 4.8kilobases (kb). AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in numerous species. Its single-stranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging, while Cap expression gives rise to the viral capsid proteins (VP; VP1/VP2/VP3), which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization [6]. It is estimated that the viral coat is comprised of 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1:1:10 (VP1:VP2:VP3) [6]. The aap gene encodes the assembly-activating protein (AAP) in an alternate reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly [7]. While AAP is essential for nucleolar localization of VP proteins and capsid assembly in AAV2, the subnuclear localization of AAP varies among 11 other serotypes recently examined, and is nonessential in AAV4, AAV5, and AAV11 [8].

Although there is much more to the biology of wild-type AAV, much of which is not fully understood, this is not the form that is used to generate gene therapeutics. Recombinant AAV (rAAV), which lacks viral DNA, is essentially a protein-based nanoparticle engineered to traverse the cell membrane, where it can ultimately traffic and deliver its DNA cargo into the nucleus of a cell. In the absence of Rep proteins, ITR-flanked transgenes encoded within rAAV can form circular concatemers that persist as episomes in the nucleus of transduced cells [9]. Because recombinant episomal DNA does not integrate into host genomes, it will eventually be diluted over time as the cell undergoes repeated rounds of replication. This will eventually result in the loss of the transgene and transgene expression, with the rate of transgene loss dependent on the turnover rate of the transduced cell. These characteristics make rAAV ideal for certain gene therapy applications. Following is an overview of the practical considerations for the use of rAAV as a gene therapy agent, based on our current understanding of viral biology and the state of the platform. The final section provides an overview for how rAAV has been incorporated into clinical-stage gene therapy candidates, as well as the lessons learned from those studies that can be applied to future therapeutic opportunities.

The main point of consideration in the rational design of an rAAV vector is the packaging size of the expression cassette that will be placed between the two ITRs. As a starting point, it is generally accepted that anything under 5kb (including the viral ITRs) is sufficient [10]. Attempts at generating rAAV vectors exceeding packaging cassettes in excess of 5kb results in a considerable reduction in viral production yields or transgene recombination (truncations) [11]. As a result, large coding sequences, such as full-length dystrophin, will not be effectively packaged in AAV vectors. Therefore, the use of dual, overlapping vector strategies (reviewed by Chamberlain et al.) [12], should be considered in these cases. An additional consideration relates to the biology of the single-stranded AAV-delivered transgenes. After delivery to the nucleus, the single-stranded transgene needs to be converted into a double-stranded transgene, which is considered a limiting step in the onset of transgene expression [13]. An alternative is to use self-complementary AAV, in which the single-stranded packaged genome complements itself to form a double-stranded genome in the nucleus, thereby bypassing that process [13, 14]. Although the onset of expression is more rapid, the packaging capacity of the vector will be reduced to approximately 3.3kb [13, 14].

AAV2 was one of the first AAV serotypes identified and characterized, including the sequence of its genome. As a result of the detailed understanding of AAV2 biology from this early work, most rAAV vectors generated today utilize the AAV2 ITRs in their vector designs. The sequences placed between the ITRs will typically include a mammalian promoter, gene of interest, and a terminator (Fig.). In many cases, strong, constitutively active promoters are desired for high-level expression of the gene of interest. Commonly used promoters of this type include the CMV (cytomegalovirus) promoter/enhancer, EF1a (elongation factor 1a), SV40 (simian virus 40), chicken -actin and CAG (CMV, chicken -actin, rabbit -globin) [15]. All of these promoters provide constitutively active, high-level gene expression in most cell types. Some of these promoters are subject to silencing in certain cell types, therefore this consideration needs to be evaluated for each application [16]. For example, the CMV promoter has been shown to be silenced in the central nervous system (CNS) [16]. It has been observed that the chicken -actin and CAG promoters are the strongest of these constitutive promoters in most cell types; however, the CAG promoter is significantly larger than the others (1.7kb vs. 800bp for CMV), a consideration to take into account when packaging larger gene inserts [15].

Schematic representation of the basic components of a gene insert packaged inside recombinant AAV gene transfer vector. AAV adeno-associated virus, ITR inverted terminal repeat

Although many therapeutic strategies involve systemic delivery, it is often desirable to have cell- or tissue-specific expression. Likewise, for local delivery strategies, undesired systemic leakage of the AAV particle can result in transduction and expression of the gene of interest in unwanted cells or tissues. The muscle creatine kinase and desmin promoters have been used to achieve high levels of expression, specifically in skeletal muscle, whereas the -myosin heavy chain promoter can significantly restrict expression to cardiac muscle [15, 17]. Likewise, the neuron-specific enolase promoter can attain high levels of neuron-specific expression [18, 19]. Often is the case, systemic delivery of AAV results in a significant accumulation in the liver. While this may be desirable for some applications, AAV can also efficiently transduce other cells and tissues types. Thus, in order to restrict expression to only the liver, a common approach is to use the 1-antitrypsin promoter [20, 21]. Finally, there are now technologies that have the ability to generate novel, tissue-specific promoters, based on DNA regulatory element libraries [22].

Over the course of the past 1015years, much work has been done to understand the correlation between codon usage and protein expression levels. Although bacterial expression systems seem to be most affected by codon choice, there are now many examples of the effects of codon engineering on mammalian expression [23]. Many groups have developed their own codon optimization strategies, and there are many free services that can similarly provide support for codon choice. Codon usage has also been shown to contribute to tissue-specific expression, and play a role in the innate immune response to foreign DNA [24, 25]. With regard to the gene of interest, codon engineering to support maximal, tissue-specific expression should be performed.

Additionally, terminator/polyadenylation signal choices, the inclusion of post-transcriptional regulator elements and messenger RNA (mRNA) stability elements, and the presence of microRNA (miRNA) target sequence in the gene cassette can all have effects on gene expression [26]. The human factor IX 3 UTR, for example, was shown to dramatically increase factor IX expression in vivo, especially in the context of additional cis regulatory elements [27]. Likewise, synthetic miRNA target sequences have been engineered into the 3 UTR of AAV-delivered genes to make them susceptible to miRNA-122-driven suppression in the liver [28]. Although there is much known about these individual components that needs to be considered when designing an AAV vector, the final design will most likely need to be determined empirically. It is not yet possible to know how a particular design will function by just combining the best elements together based on published reports, therefore considerable trial and error will eventually be required for deciding on the final construct. In addition, one also needs to consider the differences between in vitro and in vivo activity. Although it is possible to model rAAV expression in rodents, there is still significant concern about the translatability to humans.

AAV has evolved to enter cells through initial interactions with carbohydrates present on the surface of target cells, typically sialic acid, galactose and heparin sulfate [29, 30]. Subtle differences in sugar-binding preferences, encoded in capsid sequence differences, can influence cell-type transduction preferences of the various AAV variants [3133]. For example, AAV9 has a preference for primary cell binding through galactose as a result of unique amino acid differences in its capsid sequence [34]. It has been postulated that this preferential galactose binding could confer AAV9 with the unique ability to cross the bloodbrain barrier (BBB) and infect cells of the CNS, including primary neurons [35, 36].

In addition to the primary carbohydrate interactions, secondary receptors have been identified that also play a role in viral transduction and contribute to cell and tissue selectivity of viral variants. AAV2 uses the fibroblast/hepatocyte growth factor receptor and the integrins V5 and 51; AAV6 utilizes the epidermal growth factor receptor; and AAV5 utilizes the platelet-derived growth factor receptor. Recently, an uncharacterized type I membrane protein, AAVR (KIAA0319L), was identified as a critical receptor for AAV cell binding and internalization [37].

As a result of these subtle variations in primary and secondary receptor interactions for the various AAV variants, one can choose a variant that possesses a particular tropism and preferentially infects one cell or tissue type over others (Table). For example, AAV8 has been shown to effectively transduce and deliver genes to the liver of rodents and non-human primates, and is currently being explored in clinical trials to deliver genes for hemoglobinopathies and other diseases [38]. Likewise, AAV1 and AAV9 have been shown to be very effective at delivering genes to skeletal and cardiac muscle in various animal models [3946]. Engineered AAV1 is currently being explored as the gene transfer factor in clinical trials for heart failure, and has been approved for the treatment of lipoprotein lipase deficiency [47]. However, although different AAV vectors have been identified that preferentially transduce many different cell types, there are still cell types for which AAV has proven difficult to transduce.

Selected AAV vectors, known receptors, and known tropisms

With the strong desire to utilize AAV to deliver genes to very selective cell and tissue types, efforts to clone novel AAV variants from human and primate tissues have identified a number of unique capsid sequences that are now being studied for tropism specificities [48]. In addition, recombinant techniques involving capsid shuffling, directed evolution, and random peptide library insertions are being utilized to derive variants of known AAVs with unique attributes [4951]. In vivo-directed evolution has been successfully used to identify novel AAV variants that preferentially transduce the retinal cells of the eye, as well as other cell populations, including those in the CNS [50, 52, 53]. In addition, these techniques have been employed to identify novel AAV variants with reduced sensitivities to neutralizing antibodies (NAbs) [5457].

Alternatively, other investigators have inserted larger binding proteins into different regions of AAV capsid proteins to confer selectivity. For example, DARPins (designed ankyrin repeat proteins), portions of protein A, and cytokines, have all been engineered into the capsid of AAV for the purpose of greater cell specificity and targeting [58, 59]. Employing this concept, others have been able to selectively target AAV to tumors and CD4+ T cells, as examples of engineered tropism [60, 61].

As we continue to learn more about the biology of AAV with regard to the mechanisms involved in membrane translocation, endosomal escape, and nuclear entry, we will undoubtedly find opportunities to engineer unique properties into viral vectors through modulating one or more of these functions. For example, it has been hypothesized that surface-exposed serine and tyrosine residues could be phosphorylated upon viral cell entry, resulting in their ubiquitination and proteolytic degradation [6264]. Studies have shown that mutation of tyrosine to phenylalanine, which prevents this phosphorylation, results in dramatically improved transduction efficiencies [63]. Similar efforts have been made in attempts to limit the effects of NAbs, as discussed below.

The choice of a particular AAV to use as a gene transfer vector is heavily reliant on several critically important criteria: (1) which cell/tissue types are being targeted; (2) the safety profile associated with the delivered gene; (3) the choice of systemic versus local delivery; and (4) the use of tissue-specific or constitutively active promoters. As one gives careful consideration to these selection criteria, it is possible to narrow the choices of which AAVs (natural or engineered) to profile. Alternatively, one can begin the path of exploring fully engineered versions of AAV for truly selective cell targeting and optimized transduction. Because our understanding of AAV biology is in relative infancy, many of these efforts will remain empirical for quite some time as optimization for one activity could have a negative impact on another. Nonetheless, the future looks promising for this highly adaptable platform.

One of the appealing aspects of using rAAV as a gene transfer vector is that it is composed of biomolecules, i.e. proteins and nucleic acids. Fortunately, a full-package virus lacks engineered lipids or other chemical components that could contribute to unwanted toxicities or immunogenicities that may not be predictable or fully understood. In general, AAV has been shown to be less immunogenic than other viruses. Although not completely understood, one possible reason for this may hinge on the observation that certain AAVs do not efficiently transduce antigen-presenting cells (APCs) [65]. Additionally, unlike previous viral delivery strategies, rAAV does not contain any viral genes, therefore there will be no active viral gene expression to amplify the immune response [66]. Although AAV has been shown to be poorly immunogenic compared with other viruses (i.e. adenovirus), the capsid proteins, as well as the nucleic acid sequence delivered, can trigger the various components of our immune system. This is further complicated by the fact that most people have already been exposed to AAV and have already developed an immune response against the particular variants to which they had previously been exposed, resulting in a pre-existing adaptive response. This can include NAbs and T cells that could diminish the clinical efficacy of subsequent re-infections with AAV and/or the elimination of cells that have been transduced. It should be of no surprise that the formidable challenge is how to deliver a therapeutically efficacious dose of rAAV to a patient population that already contains a significant amount of circulating NAbs and immunological memory against the virus [67]. Whether administered locally or systemically, the virus will be seen as a foreign protein, hence the adaptive immune system will attempt to eliminate it.

The humoral response to AAV is driven by the uptake of the virus by professional APCs, and their presentation of AAV capsid peptides in the context of class II major histocompatibility proteins (MHCs) to B cells and CD4+ T cells [68, 69]. This leads to plasma cell and memory cell development that has the capacity to secrete antibodies to the AAV capsid. These antibodies can either be neutralizing, which has the potential to prevent subsequent AAV infection, or non-neutralizing. Non-NAbs are thought to opsonize the viral particles and facilitate their removal through the spleen [70].

Upon entry of the virus into target cells during the course of the natural infection process, the virus is internalized through clathrin-mediated uptake into endosomes [71]. After escape from the endosome, the virus is transported to the nucleus where the ITR-flanked transgene is uncoated from the capsid [72]. The pathway and mechanism of AAV intracellular transport and processing is not fully understood, and there are quite a few areas of debate with regard to current understanding. The most current hypothesis is that following endosomal escape, capsid breakdown and uncoating occurs after subsequent nuclear translocation. However, it is thought that cytosolic ubiquitination of the intact virus can occur during transport to the nucleus [73]. This would be a critical step in directing capsid proteins to the proteasome for proteolytic processing into peptides for class I MHC presentation. This hypothesis is supported by data in which proteasome inhibitors, or mutations in capsid residues that are sites for ubiquitination, can limit class I presentation and T-cell activation [7376]. However, apparent differences have been observed for T-cell activation to different AAV variants with significant sequence identity. At this time, it is unclear whether this is due to subtle capsid sequence differences and susceptibility to MHC I presentation or differential cellular processing that is innate to the different AAV variants, or simply due to contaminants in vector preparations [76].

In addition to an adaptive immunological reaction to the capsid of AAV, the transgene can elicit both an adaptive and an innate response. If the transgene encodes a protein that can be recognized as foreign, it too can generate a similar B- and T-cell response. For example, in replacement therapy applications in which the protein to be replaced is the consequence of a null genotype, the immune system will have never selected against precursor B and T cells to that protein [70, 77]. Likewise, if the transgene is an engineered variant, the engineered sequence can be recognized as foreign. Even the variable regions of antibodies can activate an adaptive response that can result in deletion of target cells that are expressing transgene as a result of AAV delivery. Finally, a transgene with a significant number of CpG dinucleotides can activate innate responses through toll-like receptor (TLR) molecular pattern receptors [78].

Pre-existing immunity to AAV, especially the presence of circulating NAb, can have a dramatic effect on AAV clinical efficacy. To date, this represents one of the biggest therapeutic challenges to the use of systemically delivered AAV, and is thought to be one of the factors in early clinical failures [79]. Pre-existing immunity to AAV can often be overcome by selecting a particular AAV variant that has not circulated throughout the human population, and, therefore, does not have any memory responses elicited against it, including NAbs and T cells [80]. Additionally, some of the AAV evolution technologies discussed above have been used to identify AAVs that are resistant to the effects of NAbs [50, 57]. Although not optimal, it is possible to prescreen subjects for the presence of NAbs to the particular AAV variant to be used. In addition, the impact of this immunological response can sometimes be minimized by the particular route of administration employed for the particular therapeutic strategy, as discussed in Sect. 6 [80].

Like most biotherapeutics, AAV needs to be produced in a living system (Fig.). The parallels with recombinant antibody production during the 1990s and 2000s, with regard to the upstream challenges of robust production levels, are important to understand where the industry currently is, and where we need to strive to be.

Overview of AAV production/purification. Cell platform: HEK-293T, Sf9, or other suitable cell system can be grown on a small scale on 150mm tissue culture-treated culture dish, hyperflasks, or shake flasks. Cells are then transfected with adenovirus helper virus, rep/cap, and ITR-transgene plasmids for 293T, or infected with baculovirus for Sf9. Producer lines with integrated expression of rep/cap and ITR-transgene can be infected with adenovirus and grown to scale. Scale-up: For larger-scale culture volumes, virus can be produced in roller bottles, continuous perfusion, or WAVE Bioreactor systems. Purification/polishing: Affinity or heparin chromatography are optimal for isolation of virus from culture supernatants with or without cell pellet harvesting. Benzonase/DNAse treatment of eluted virus is required for removal of extraviral DNA contamination, followed by anion-exchange chromatography to fractionate empty vs. full AAV particles. QC/release: Upper left of far right panel: image depicts a silver stain analysis of culture FT next to affinity/anion exchange purified AAV (pure). The three bands represent the viral capsid proteins VP1, VP2, and VP3. Upper right of far right panel: Dynamic light scattering analysis of purified AAV1 indicates a uniform particle distribution of approximately 2530nM. Bottom half of far right panel: Analytical ultracentrifugation can resolve the proportion of empty vs, full particles of purified material. Additional assays that should be employed are digital drop polymerase chain reaction for determining titer in GC/mL, cryo or transmission electron microscopy for visual representation of purified particles, endotoxin testing, and other assays to evaluate the presence of residual host-cell protein contamination. AAV adeno-associated virus, FT flow-through, GC genome copies, rep/cap replication/capsid, QC quality control

Current methods to produce rAAV are still expensive despite years of research (Table). The most widely used platform for producing rAAV involves transfecting HEK293 cells with either two or three plasmids; one encoding the gene of interest, one carrying the AAV rep/cap genes, and another containing helper genes provided by either adeno or herpes viruses [6]. While most robust production rates have been achieved with adherent cells in either roller bottles or cell stacks, similar rates are now achievable in suspension-adapted HEK293 cells (Table). Production rates of approximately 105 genome copies (GC)/cell are now common, resulting in 1014 GC/L [81]. While this has proven to be sufficient to support early clinical trials, and could supply marketed product for small patient population indications, the deficiencies in scalability with this platform are a significant limitation [82, 83]. As one could surmise, successfully delivering three plasmids to one cell is a relatively inefficient process. For larger-scale manufacturing efforts, transient delivery of plasmid requires excess quantities of DNA, adding to the overall cost of production and purification. Moreover, transient delivery of rep/cap genes in the presence of helper genes can also contribute to product heterogeneity, including AAV vectors lacking a transgene. These empty capsids represent a significant proportion of virus produced in transient transfection assays. Thus, it is critically important to develop robust analytical quality control (QC) methods that are able to distinguish between these viral variants in order to ensure similarities between production lots [82, 83].

Current manufacturing platforms being employed to generate rAAV for clinical use

In three other AAV manufacturing platforms, one or more genetic components for the AAV manufacturing has been integrated into the genome of mammalian or insect production cell lines. While most viral helper genes needed for AAV production cannot be stably transfected, the adenoviral E1a and E1b genes are exceptions. These genes have been used to transform HEK293 cells, however they induce expression of the AAV rep gene, which is toxic to mammalian and insect cells [84, 85]. Hence, two different approaches have been used to develop mammalian cell lines. The first uses co-infection of BHK cells with two replication-defective HSVs engineered to encode the ITR-flanked transgene and the rep/cap genes. The second is based on stable producer cell lines in HeLa cells carrying the ITR-flanked transgene and the rep/cap genes. Rep proteins are not expressed in these cells since HeLa carries no adenoviral genes. However, infection with wild-type adenovirus is required for AAV production. The inclusion of replication-competent viral agents into a production process is a concern that needs to be addressed and also requires additional steps during the downstream processing [82, 83].

More recently, the Sf9 insect cell system in combination with baculovirus infection has been utilized to produce bulk quantities of rAAV. In this system, two or three baculovirus particles may be used to infect the Sf9 cells and initiate AAV production. In one example, one virus contains the rep gene, a second contains the cap gene, and the final virus carries the ITR-flanked gene of interest. In an alternative system, the Sf9 cells can be engineered to have the ITR-flanked gene of interest integrated into their genome, upon which production is initiated with only two baculovirus preps [81, 82]. A further improvement has recently been shown whereby the rep/cap genes are stably integrated into the Sf9 cell line genome, but are under the control of a promoter/enhancer that is induced by subsequent baculovirus infection. In this system, infection can occur, with only one baculovirus containing the ITR-flanked gene of interest, simplifying the system significantly [86, 87].

Production levels of approximately 105 GC/cell and 1015 GC/L have routinely been achieved with these Sf9 systems. Because of their ease of manipulation and their ability to grow to very high cell densities, the Sf9 system is rapidly becoming the platform of choice for AAV manufacturing. Concerns regarding baculovirus instability and differences in post-translational modifications between mammalian and insect cell systems are now beginning to be understood and controlled. These concerns are offset by the fact that baculovirus cannot efficiently infect mammalian cells which makes it inherently safer then other viral-based production systems [8183, 86, 87].

Unlike antibody manufacturing that relied on a single protein A-based purification platform early in the development of the downstream process, AAV is still rapidly evolving in that area. The products of an AAV production run will contain not only cellular debris (protein/lipids/nucleic acids) but also two main populations of AAV particles: particles that contain (full capsids) or those lacking (empty capsids) the ITR-flanked transgene. Although still widely debated in the field, the presence of empty capsids represents another contaminant that must be removed or controlled. Initial attempts to separate these two populations originally relied on the cumbersome and non-scalable method of density ultracentrifugation. In addition to the scalability issue, there are also concerns about the physiochemical effects of this method on the particles. Regardless, this method is still employed by many organizations as either a primary or secondary step in AAV purification [83].

Current technologies utilizing various affinity resins and/or ion exchange chromatography are being adopted by the industry. As mentioned above, AAV uses cell membrane-associated carbohydrates as the primary cell receptor for transduction. This affinity for carbohydrates can be exploited as an initial capture step in AAV purification. Indeed, heparin columns are frequently used in many downstream processing steps for AAV [88]. However, because of the lack of specificity, alternative affinity columns based on AAV-specific binding proteins such as scFvs and antibody single domains from llamas (camelids) have started to dominate the field. Improvements in generating these AAV-specific resins confers many advantages in downstream purification. These resins have the ability to bind to more than one AAV variant, have very high binding capacities (>1014GC/mL resin), and are stable against harsh clean-in-place and regeneration methods, making them suitable for use multiple times. Some of these commercial resins are already Good Manufacturing Practice (GMP) compliant, making them ideal for downstream manufacturing at commercial scales. Polishing steps using anion exchange chromatography are now routinely included after affinity capture steps, and can efficiently separate full capsids from empty capsids [8992].

As with any new therapeutic platform, and, again, similar to antibody-based therapeutic evolution, details on product specification and regulatory requirements are still evolving. With still very limited clinical experience, the impact of empty particles, host-cell impurities, post-translational modifications from different production platforms, fidelity of the packaged transgene, capsid ratio integrity, and probably many other specifications are still not known. However, over time, and as more clinical experience is gained, the field will be able to better relate these details to product performance and safety [83].

The use of rAAV as a delivery vector for gene therapies has been rapidly gaining interest over the past 35years. As approvals begin to increase (see Sect.6), efforts to optimize and maximize clinical manufacturing technologies will see a burst of activity. This will most likely mirror what occurred with antibody therapeutics in the 1990s and 2000s, in which early technologies were quickly overcome by next-generation technologies, resulting in significant cost savings and increased clinical supplies.

AAV has been shown to be a very stable vector able to withstand wide temperature and pH changes with little to no loss in activity [93]. To date, the only limitation seems to be the concentration with which it can be formulated, currently maximized around 51013 particles per milliliter [83]. With the resurgence in clinical use, this formulation limit will most likely be overcome in the near future. However, the robust stability of these vectors provides ample opportunities to attempt different routes of administration and specialized delivery strategies (Table).

Selected examples of more than 50 clinical candidates employing rAAV

Other than the European Medicines Agency (EMA)-approved AAV-based product alipogene tiparvovec (Glybera), the most advanced current clinical trial using AAV is sponsored by Spark Therapeutics and utilizes local injection of AAV2 into the eye for inherited retinal diseases (voretigene neparvovec-RPE65) (Table) [94]. Phase III studies have just been completed on this candidate and a Biologics License Application (BLA) submission is expected this year. This type of local delivery has proven to be safe and efficacious, but requires specialized surgical techniques and/or devices to deliver the vector [94, 95]. Similar strategies are being conducted by Applied Genetic Technologies Corporation (AGTC), targeting X-linked retinoschisis and achromatopsia, X-linked retinitis pigmentosa, and age-related macular degeneration. These programs are at various stages of development, with the most advanced for X-linked retinoschisis and achromatopsia in phase I safety studies (http://www.AGTC.com) (Table).

Several clinical trials are being run in which systemic administration is being used to target the liver, a tissue that is readily accessible through this route of administration and a tissue type that is readily transduced by many well-understood AAV variants [96]. These trials are mostly for monogenic, inherited diseases, in which the goal is gene replacement for defective genes, including those mutated in hemophilia A and B. Currently, these trials are in phase I/II, and are sponsored by academic groups, as well as biopharmaceutical companies such as Spark Therapeutics (SPK-9001, SPK-8011), Sangamo Therapeutics (SB-525), UniQure (AMT-060), Dimension Therapeutics (DTX101, DTX201), and Biomarin (BMN 270) (Table) [97]. Unlike local administration to the eye, which is considered an immune-privileged site that might not be affected by the existence of NAbs, systemic administration will require patient stratification for patient NAb levels. In addition, the possibility for re-administration becomes very difficult, should the need arise [80]. Although rare, there have been reports of rAAV vector integration into animal model genomes with subsequent genotoxicities [98, 99]. In addition, AAV genome sequences have been found in human hepatocellular carcinoma samples near known cancer driver genes, although at a low frequency [100]. There is an ongoing debate on these findings regarding cause and effect, and mouse/human translation. Regardless, hepatocellular, as well as other tissue genotoxicity, will need to be monitored in the course of AAV clinical development.

Another common delivery strategy is direct intramuscular injections. The only approved AAV gene therapy in Europe (Glybera) is an AAV1 encoding the gene for lipoprotein lipase deficiency [47, 101]. Skeletal muscle has been shown to be a target tissue type that is efficiently transduced by many AAV variants [39]. Once transduced, the muscle cells serve as a production site for protein products that can act locally or systemically, as is the case with Glybera. As a result of the low cellular turnover rate of the muscle cells, the transduced AAV gene product will be maintained in these cells as an episome for years, as has been shown in many studies in non-human primates [39]. Consequently, a single-dose regimen of an intramuscularly-delivered product may never need to be readministered unless there is significant damage or immune clearance of the transduced cells. This strategy is also being employed by Adverum and AGTC for 1-antitrypsin deficiency, as well as for certain muscular dystrophies (Table) [97].

Direct CNS administration is being utilized for Parkinsons disease, as well as various inherited diseases such as Batten disease, Canavan disease, and mucopolysaccharidosis (MPS) IIA and IIB, as well as MPS IIIa and MPS IIIb (Sanfilippo syndromes type A and type B, respectively). Phase I/II studies for these diseases using a variety of AAV variants, including AAV2, AAVrh10, and AAV9, are currently ongoing by various academic groups and biopharmaceutical companies, such as Abeona Therapeutics (ABO-101, ABO-102, ABO-201, ABO-202) [97, 102, 103]. Delivery strategies range from direct intraparenchymal administration into particular areas of the brain, intracerebroventricular, and cisternal and lumbar intrathecal routes [102]. The decision on the best route of administration is intimately related to the disease and affected areas. For example, for Parkinsons disease, according to our current understanding of disease pathogenesis and therapeutic strategies, direct injection into the putamen, substantia nigra or striatum is thought to be required. Similarly, for diseases that affect larger areas of the brain, such as Canavan disease or MPS, direct injection into the cerebellum is thought to be most beneficial [102, 103].

Alternatively, administration directly into the cerebrospinal fluid through an intrathecal route can result in wide CNS biodistribution, which is thought to be necessary for diseases such as spinal muscular atrophy (SMA) and Alzheimers disease [102106]. An alternative to cerebral spinal fluid (CSF)-based routes is the use of systemic administration of AAV variants that have been shown to cross the BBB. AAV9 has been shown to transcytosis across the BBB and transduce large sections of the CNS [36, 104, 107, 108]. This approach is currently being explored in the clinic for the treatment of SMA by AveXis (AVXS-101).

Neurodegenerative diseases represent a particular devastating health problem for which there is significant unmet medical need. These diseases of the CNS have proven to be very difficult to treat as a result of our poor understanding of their etiology and difficulty getting efficacious agents across the BBB. With regard to Alzheimers disease, although there is still some disagreement in the field, idiopathic amyloid plaque formation or generation of neurofibrillary tau tangles (NFTs), both of which are thought to be neurotoxic, are still the prevailing hypotheses behind the mechanism of many of these neuropathologies. Attempts to clear these plaques with plaque-specific antibodies have shown signs of limiting this process in animals and early-stage clinical trials [109, 110]; However, larger studies have all shown to be inconclusive at best, or failures at worst. It is unclear if these failures were because the plaque hypothesis is wrong, or if there was inefficient CNS exposure to the antibody therapeutic [110, 111]. Alternative strategies taking advantage of the safety and persistence of AAV would utilize either local administration of antibody-encoding AAVs directly to the CNS, or systemic delivery of AAVs that can cross the BBB, resulting in significantly higher CNS exposure levels of the antibody [112].

Local delivery of AAV to cardiac muscle for heart failure has been attempted in various clinical trials. In one case, Celladon failed in their attempt to deliver SERCA2A directly to the heart, and, in a second case, there is an ongoing program sponsored by UniQure to deliver S100A directly to the heart that is currently still in preclinical development [46, 113115]. Although it is not thoroughly clear why Celladon failed in the clinic, and why one would expect UniQure/BMS to succeed, there are significant differences in the delivery methods used by the two programs and the target gene delivered. Celladon used intracoronary infusion to deliver their AAV1 SERCA2A gene product, whereas UniQure is using retroinfusion and left anterior descending (LAD) coronary occlusion [41, 115]. This procedure is thought to better localize and restrict the delivered AAV9 S100A gene product to better target the heart tissue of interest. The reality of this suspected benefit will be realized in the clinic in the coming years.

Aerosolized AAV for inhaled pulmonary delivery was utilized in some of the earliest trials for cystic fibrosis (CF). Although none of these trials resulted in significant benefit or showed much of a pharmacodynamic response, they did help to show the safety of AAV when administered via this route [116118]. More importantly, the pathophysiology of CF, molecular biology of the CF transmembrane conductance regulator (CFTR) gene, and the target cell population for this type of indication exposed some key considerations when using AAV [117]. Congestion of the airways in these patients can limit AAV biodistribution after delivery, thus attenuating robust transduction [118]. In addition, the CFTR gene is over 4kb in size, putting it at the upper limit of the packaging capacity of AAV after also considering a required promoter and terminator. Finally, CFTR is expressed by the submucosal glands, which may be difficult to target efficiently [116, 117]. Nonetheless, these early efforts proved that AAV can safely deliver genes to the lung, which might be an ideal strategy for other diseases, such as influenza and other infectious diseases of the lung [119].

The field is just beginning to explore localized delivery of AAV for gene therapy applications. The stability of the virus and broad tropism for many different cell and tissue types make them ideal for most applications. There appears to be at least one AAV variant option for every tissue type of interest, with engineering and novel AAV discovery efforts sure to identify and create AAV variants with very specialized functions on demand. These efforts will undoubtedly result in new therapeutic strategies for many new indications.

The transfer of genes and other nucleic acids into cells has been a research tool in the laboratory for more than four decades. However, it was our growing understanding of the genetic components underlying certain diseases that has driven the search for true gene therapies. Progressively, research in other areas have identified other potential opportunities in which gene delivery could be applied therapeutically. In addition, limitations with current small molecule and protein therapeutic platforms have also driven the search for alternative therapeutic platforms that accommodate those limitations [120, 121]. Gene therapies accommodate all of those limitations, especially around target accessibility. As a result, the search for safe and effective gene delivery technologies has been a major focus in pharmaceutical research and development, and will hopefully represent a paradigm shift in how we approach disease-state intervention.

AAV was discovered over 50years ago and has since become one of the leading gene delivery vectors in clinical development. As a result of its unique biology, simple structure, and no known disease associations, AAV could become the vector of choice for most gene therapy applications. Gene therapy using rAAV has been demonstrated to be safe and well-tolerated in virtually every clinical setting in which it has been used. These studies, along with basic research on its biology, have revealed many facets of this vector that can be applied to future efforts.

Among the critical parameters to be considered are vector design, capsid selection, desired target cell and tissue type, and route of administration. The transgene to be delivered optimized for expression, the right AAV variant with an appropriate capsid for target cell transduction and immunoreactivity profile, and the appropriate delivery approach to maximize target tissue exposure while limiting off-tissue exposure are key focal points for AAV-based therapies.

All of these variables will be dictated by the overall therapeutic strategy which will be influenced by our understanding of the pathobiology of the disease to be treated. Will the transgene have the desired effect? Is the target cell driving the disease state? Is the turnover rate of the target cell high, requiring repeat dosing? This cannot be emphasized enough; without a strong understanding of the mechanisms driving the disease state, it will not be possible to design, discover, and develop the right gene therapeutic. Better designed trials, optimized vector construction, and novel AAV variants will certainly result in future regulatory approvals and improvements on patient outcomes and health.

Michael F. Naso, Brian Tomkowicz, and William L. Perry III are employees of Janssen Research and Development. William R. Strohl has no conflicts of interest to declare.

No funding was received for the preparation of this review.

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Adeno-Associated Virus (AAV) as a Vector for Gene Therapy

Foundation Fighting Blindness is a driving force in advancing retinal gene therapies into clinical trials.

Growth in the clinical and commercial development of gene therapies for retinal degenerative diseases has been explosive over the past decade. The rapid expansion of the field has been led by dramatic vision restoration for virtually blind patients made possible by voretigene neparvovec-rzyl(Luxturna; Spark Therapeutics), which the FDA approved in December 2017. It is the first gene therapy for the eye or any inherited retinal disease to cross the regulatory finish line in the United States. Developed by gene therapy pioneer Jean Bennett, MD, PHD, for children and adults with Leber congenital amaurosis (LCA) or retinitis pigmentosa (RP) caused by biallelic RPE65 mutations, the therapy provided immediate and impressive vision improvements in a phase 1/2 clinical trial at Childrens Hospital of Philadelphia. Those initial results, reported in 2008, sent a strong signal to investigators and biotechnology companies that gene therapy could be a powerful modality for treating retinal diseases.

Now, dozens of companies are in the retinal gene therapy development space and more than 20 clinical trials for genetic therapies are underway for patients with a broad range of retinal degenerative diseases. These include RP, LCA, choroideremia, achromatopsia, and the dry and wet forms of age-related macular degeneration (AMD).

The Foundation Fighting Blindness, the worlds leading private funder of retinal degenerative research, has played a pivotal role in advancing retinal gene therapies into clinical development. As an early funder of the modality for myriad retinal diseases, the foundation began investing in RPE65 gene therapy research back in the mid-1990s and ultimately invested $10 million in the research that eventually led to voretigene neparvovec.

We were very early adopters, recognizing that the retina was an ideal gene therapy target. Its a small, accessible piece of tissue, and many conditions that affect the retina are monogenic. We knew that if we could directly address the patients mutated gene by augmenting or modifying its activity, we had an excellent opportunity to save and restore vision, said Benjamin Yerxa, PhD, chief executive officer at the foundation. Furthermore, we see gene therapys potential for gene-agnostic applications such as neuroprotection and optogenetics to help a broad range of patients, regardless of the mutated gene causing their vision loss.

The foundation currently funds a broad range of gene therapies and other treatment modalities at both early and late stages of development. Its RD (Retinal Degeneration) Fund, a venture philanthropy fund with nearly $120 million in commitments, was launched in 2018 to help investigators and start-up companies move their emerging therapies into and through early-stage clinical trials. The funds goal is to attract additional investments from pharmaceutical and biotechnology companies to fund the more expensive, late-stage trial phases. Furthermore, all returns on the funds investments are put back into the foundation to support additional research and investments.

The ultimate goal of the RD Fund is to get more treatments across the finish line and out to patients. We cant afford to fund late-stage clinical research, which often costs a hundredmillion dollars or more, but we can afford the earlier-stage research to attract those major investments from biotechs and big pharma, said Yerxa.

In fall 2021, the RD Fund took the bold step of launching its own company, Opus Genetics, to develop gene therapies for orphan retinal diseases, those rare conditions that werent being addressed by other companies. Opus $19 million in seed financing included investments from the Manning Family Foundation and Bios Partners. Its first 3 targets are for LCA caused by mutations in LCA5, RDH12, and NMNAT1. The LCA5 and RDH12 therapies were developed preclinically by Opus cofounder Bennett and licensed from the University of Pennsylvania. The NMNAT1 treatment was developed in the laboratory by cofounder Eric A. Pierce, MD, PhD, and licensed from Harvard Medical Schools Massachusetts Eye and Ear. Opus plans to launch a clinical trial for LCA5 by the end of 2022. In April 2022, the company signed a collaboration agreement with Resilience to provide manufacturing services for its gene therapy pipeline.

In October 2020, the RD Fund realized its first financial win when Novartis acquired Vedere Bio for around $280 million. In 2019, the fund had helped launch Vedere Bio to advance an optogenetic therapy developed by its scientific cofounders John G. Flannery, PhD, and Ehud Isacoff, PhD, from the University of California, Berkeley. The investigators approacha gene-agnostic gene therapyprovides potential vision restoration for patients who have lost all their photoreceptors to a condition such as RP by delivering a gene that expresses a light-sensing green cone opsin in surviving ganglion cells. In essence, the treatment enables ganglion cells to work like a back-up system for lost photoreceptors. The approach holds promise for restoring vision for patients who are completely or nearly blind, regardless of the mutation causing their disease. A new incarnation of the company, Vedere Bio II, was subsequently launched after the Novartis acquisition to continue development of other retinal gene therapies.

Also in 2020, the RD Fund invested in the gene therapy start-up Atsena Therapeutics, which reported early, encouraging vision improvements for 3 patients in a phase 1/2 clinical trial for its LCA (GUCY2D mutations) gene therapy. Cofounded by Shannon E. Boye, PhD, and Sanford L. Boye, MS, both of the University of Florida in Gainesville, Atsena also has preclinical gene therapy programs for X-linked retinoschisis and Usher syndrome type 1B.

SparingVision, another RD Fund investment, is planning to launch a clinical trial in 2022 for its gene-agnostic, cone-preserving therapy for patients with RP, Usher syndrome, and related conditions. Nearly 2 decades ago, Jos-Alain Sahel, MD, and Thierry Lveillard, PhD, investigators from the Institut de la Vision in Paris, France, identified a protein secreted by rod photoreceptors that is critical to the survival of cones. Aptly named rod-derived cone-viability factor, it is the protein expressed by SparingVisions cone-preserving gene therapy. The company is also developing a gene therapy that restores light sensitivity to cones that have lost their ability to process light due to advanced forms of RP, Usher syndrome, and related diseases.

The RD Funds other gene-related therapy investments include SalioGen Therapeutics, whose Saliogase technology seamlessly inserts new DNA of any size (eg, the Stargardt disease gene ABCA4) into precise, defined genomic locations. The fund also invests in ProQR Therapeutics, which has an RNA therapy in a phase 2/3 clinical trial for individuals with Usher syndrome 2A and nonsyndromic RP caused by mutations in exon 13 of the USH2A gene.

We are off to a great start with our investments and working to continue to expand our portfolio with the strategy of investing in strong science being developed by well-managed companies, said Yerxa. I think our coinvestors recognize and appreciate our commitment to making every shot on goal really count.

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Gene therapy: Where the action is for retinal diseases - Modern Retina

Dive Brief:

Despite significant setbacks that have left the fate of its eye gene therapy in doubt and shares trading near all-time lows, Adverum hasnt given up.

The company is one of a few gene therapy makers aiming to develop a one-time treatment for diabetic macular edema and age-related macular degeneration, two common forms of vision loss that are treated with chronic injections of biologic medicines. But those drugs, like Eylea and Lucentis, are highly effective and considered safe, making the bar much higher for a gene therapy whose main goal is to improve convenience.

Adverums program was also beset by side effects the company once described as not seen before in ocular gene therapy, a combination of inflammation, vision loss and decrease in eye pressure observed in five trial participants.

Adverum stopped that trial, in diabetic macular edema, in 2021. At the time, some analysts suggested the company should attempt a reverse merger, a way for struggling biotechs to bring in new assets by combining with a privately held company seeking fast access to the public markets.

The company instead vowed to press on. Executives suggested testing a lower dose than previously planned with a different regimen of protective drugs could lead to better results in AMD. In 2021, the company noted that no cases of severe inflammation were observed in DME patients treated with a lower dose or in participants with AMD in another trial.

Adverum has since gained clearance from U.S. regulators for its new plan, a Phase 2 trial in AMD thatll test both the lowest dose evaluated in previous studies as well as one more than three-times lower. With shares trading at just over $1 apiece and equity harder to raise during the sectors downturn, Adverum has turned to cost-cutting to save money and fund the work. The savings could enable the company to get to one-year results from that trial, in 2023, without needing to raise more cash, wrote RBC analyst Luca Issi.

Yet Adverums odds remain long. A rival gene therapy from Regenxbio is already in Phase 3 testing in AMD, and pending positive results, could lead to an approval filing in 2024. The company remains a show-me story given its history, Issi wrote. Additionally, Adverums decision to turn to layoffs, rather than a partnership, may also signal limited strategic interest in the platform, he added.

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Adverum cuts jobs, restructures to give eye gene therapy another shot - BioPharma Dive

EditForce, Inc. has entered into a license agreement with Mitsubishi Tanabe Pharma Corporation (MTPC) to research, develop and commercialize potential gene therapy products for a specific target disease related to the central nervous system (CNS) by utilizing EditForces pentatricopeptide repeat (PPR) protein platform technology.

MTPC and EditForce aim to create potential novel pharmaceuticals for the specific CNS disease by utilizing the drug R&D know-how and global business experience of MTPC and the novel biotechnology of EditForce.

MTPC will acquire the exclusive right to conduct the selection of drug candidate molecules, preclinical and clinical development, manufacturing, and commercialization worldwide.

Under the terms of the agreement, EditForce will receive an upfront payment and milestone payments amounting to more than 20 billion yen ($147.3 million), depending on the development stage and commercialization progress, and royalties based on worldwide sales after the launch.

I am so delighted to reach the agreement with MTPC, which has an interest in our proprietary PPR protein platform technology, said Takashi Ono, president and CEO of EditForce.

We look forward to working closely with MTPC to develop and deliver breakthrough pharmaceutical products with our technology to patients suffering from diseases.

PPR is a protein discovered in plants that regulates gene expression by binding to DNA and RNA in a sequence-specific manner. The PPR proteins are also found in humans and yeasts, and they have similar functions.

Takahiro Nakamura and Yusuke Yagi, CTO of EditForce, have focused on the PPR proteins and elucidated the mechanism that determines sequence specificity, and established a technology for creating various PPR proteins, each of which binds to a specific target DNA or RNA sequence.

It is possible to manipulate and modify the target genome and RNA both inside and outside the cell by fusion with effector proteins.

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EditForce and Mitsubishi Tanabi Pharma to work on gene therapy for CNS - Labiotech.eu

PARIS--(BUSINESS WIRE)--Regulatory News:

Lysogene (Paris:LYS)(FR0013233475 LYS), a phase 3 gene therapy platform Company targeting central nervous system (CNS) diseases, today communicates additional preliminary data from the AAVance Phase 2/3 gene therapy trial in MPS IIIA (NCT03612869). Data will be presented at the ADVANCE 2022 Sanfilippo Community Conference held on July 7-8, 2022, and at the 3rd Annual Gene Therapy for Neurological Disorders Europe held on July 11-13, 2022.

A positive effect of LYS-SAF302 on the MPS IIIA disease biomarker heparan sulfate (HS) in the cerebrospinal fluid (CSF) was confirmed in additional subjects and at additional timepoints relative to previously communicated partial data. Statistically significant decreases of about 20% in average levels of total HS-derived oligosaccharides in the CSF relative to baseline levels were observed at 6, 12 and 24 months after dosing with LYS-SAF302. HS levels at 24 months after dosing with LYS-SAF302 (1654 497 ng/ml, mean SD, n=15) were decreased by 22% relative to baseline levels (2159 589 ng/ml, mean SD, n=16), p=0.015 by Student's t test (preliminary analysis). No statistically significant effect on serum HS levels was observed at 6, 12 or 24 months after dosing with LYS-SAF302. These results confirm the biological activity of LYS-SAF302 gene therapy treatment. They are consistent with the intraparenchymal mode of administration of LYS-SAF302, which is expected to lead to a specific decrease of HS in the brain, but not in the systemic circulation nor in other tissues, including the spinal cord.

The previous observation that treatment with LYS-SAF302 led to a transient increase in serum neurofilament light (NFL) levels, likely due to transient axonal damage caused by brain surgery, followed by a decrease below baseline levels, was confirmed in additional subjects and at additional timepoints. Moreover, a similar effect was demonstrated in the CSF. In the serum, NFL levels decreased by 33% (n=12, p=0.026) and 41% (n=16, p=0.0075) below baseline levels (113 50 pg/ml, mean SD, n=19), 18 and 24 months after dosing with LYS-SAF302, respectively. In the CSF, NFL levels decreased by 33% (n=15, p=0.025) below baseline levels (3.7 1.5 ng/ml, mean SD, n=17) 24 months after dosing with LYS-SAF302. All statistical analyses were done by Students t test and are preliminary. These results suggest that treatment with LYS-SAF302 led to a decrease in neuronal damage relative to baseline at 18 and 24 months after drug administration.

Three subjects in AAVance, treated at 10, 13 and 31 months of age, present continuous increase of cognitive, language and motor functions 24 months after dosing with LYS-SAF302, as assessed by the BSID-III (Bayleys Scales of Infant Development, Third edition). Two of these subjects have a cognitive developmental age (DA) at 24 months after dosing with LYS-SAF302 that is 5-6 months higher (41 and 42, respectively) than the highest cognitive DA (35) observed in natural history studies of MPS IIIA (Shapiro et al, 2016; Wijburg et al 2022). Remarkably, one of these subjects is homozygous for a severe mutation (deletion) and the other subject is compound heterozygous for two severe mutations (a duplication and a deletion). The third subject with continuously increasing DA at 24 months after dosing with LYS-SAF302 is a compound heterozygote for a severe mutation and a S298P mutation, which may give rise to either a classical severe or an intermediate phenotype. Longer follow-up is warranted to confirm positive evolution of development in this patient. Three other subjects, treated at 24, 30 and 31 months of age, have stable cognitive DA relative to baseline, as assessed by the BSID-III scale, and stable or continuously increasing BSID-III language and motor development scores at 24 months after dosing with LYS-SAF302. Two of these subjects have SGSH missense mutations associated with the classical severe phenotype of MPS IIIA. One subject has a severe mutation on one allele and a mutation with unknown effect on disease severity on the second allele. The fact that developmental progression or stabilization is seen in subjects with mutations associated with the classical severe disease phenotype suggests that early therapeutic intervention with LYS-SAF302 can protect children with MPS IIIA from decline of cognitive, language, and motor functions.

The AAVance trial Month 24 database lock took place as planned on 1st of July 2022. Full study results are expected by mid-September 2022, along with results from the PROVide patient reported outcome videos study. Based on this comprehensive clinical data package, the company plans to initiate discussions with regulatory authorities in the US and in Europe to determine next steps.

Preliminary data for AAVance indicates that subjects with MPS IIIA treated prior to 31 months of age not only continued with increasing developmental age, but exceeded developmental ages of any MPS IIIA subjects within the natural history cohorts. This data is highly suggestive of LYS-SAF302 efficacy in this treatment population, marking an important milestone as no treatment is currently available to slow the progression of MPS IIIA, said Raymond Wang, M.D., Director, Foundation of Caring Multidisciplinary Lysosomal Disorder Program at CHOC Childrens Specialists, Orange, CA, USA, and one of the principal investigators for the AAVance trial.

We are very pleased to confirm on a larger scale the encouraging data already observed earlier, notably stabilization or improvement in cognitive, language and motor functions in the younger patient population, even in those presenting with severe forms of the disease. We have recently locked the database of the 24-month post-treatment follow-up data. Full statistical analyses are underway with results expected in September this year. This represents a very exciting milestone as it completes years of efforts by the Lysogene team to bring a treatment to patients with MPSIIIA, a disease with a high unmet medical need. By Q3 2022, we should have the necessary elements to discuss the next steps with the regulatory authorities, said Marie Trad, M.D. Chief Medical Officer of Lysogene.

About Lysogene

Lysogene is a gene therapy Company focused on the treatment of orphan diseases of the central nervous system (CNS). The Company has built a unique capability to enable delivery of gene therapies to the CNS to treat lysosomal diseases and other disorders of the CNS. A phase 2/3 clinical trial in MPS IIIA is ongoing. An adaptive clinical trial in GM1 gangliosidosis is also ongoing. Lysogene is also developing an innovative AAV gene therapy approach for the treatment of Fragile X syndrome, a genetic disease related to autism. The Company also entered into an exclusive worldwide license agreement with Yeda, the commercial arm of the Weizmann Institute of Science, for a novel gene therapy candidate for neuronopathic Gaucher disease and Parkinson disease with GBA1 mutations. http://www.lysogene.com.

Forward Looking Statement

This press release may contain certain forward-looking statements, especially on the Companys progress of its clinical trials and cash runway. Although the Company believes its expectations are based on reasonable assumptions, all statements other than statements of historical fact included in this press release about future events are subject to (i) change without notice, (ii) factors beyond the Companys control, (iii) clinical trial results, (iv) increased manufacturing costs, (v) potential claims on its products. These statements may include, without limitation, any statements preceded by, followed by or including words such as target, believe, expect, aim, intend, may, anticipate, estimate, plan, objective, project, will, can have, likely, should, would, could and other words and terms of similar meaning or the negative thereof. Forward-looking statements are subject to inherent risks and uncertainties beyond the Companys control that could cause the Companys actual results, performance or achievements to be materially different from the expected results, performance or achievements expressed or implied by such forward-looking statements. A further list and description of these risks, uncertainties and other risks can be found in the Companys regulatory filings with the French Autorit des Marchs Financiers, including in the 2021 universal registration document, registered with the French Markets Authorities on April 19, 2022, and future filings and reports by the Company. Furthermore, these forward-looking statements are only as of the date of this press release. Readers are cautioned not to place undue reliance on these forward-looking statements. Except as required by law, the Company assumes no obligation to update these forward-looking statements publicly, or to update the reasons actual results could differ materially from those anticipated in the forward-looking statements, even if new information becomes available in the future. If the Company updates one or more forward-looking statements, no inference should be drawn that it will or will not make additional updates with respect to those or other forward-looking statements.

This press release has been prepared in both French and English. In the event of any differences between the two texts, the French language version shall supersede.

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Lysogene Provides Additional Update on AAVance Phase 2/3 Gene Therapy Clinical Trial with LYS-SAF302 in children with MPS IIIA - Business Wire