AAV vectors: selection permutations, favorable fates (2023)

Adeno-associated virus (AAV) vectors may be the best option for delivering genetic material to cells in the body. But they are far from perfect. They require large doses, carry small amounts of cargo, and can be recognized by the immune system, a safety concern. Furthermore, they behave differently in animal models such as mice and non-human primates than in humans. These complications require constant innovation and development of new strategies.

AAV vectors: selection permutations, favorable fates (1)

LogicBio Therapeutics was recently affected by discrepancies between AAV-associated immune responses in animals and humans. The well-known company had to stop the clinical trial when two participants experienced side effects. The Akouos company has just started and hopes to avoid someAvoid the pitfalls by working in one area of ​​the body, the inner ear, where the company's AAV technology has shown promise..

learn along the way

Mark A. Kay, MD, Ph.D., a professor of genetics at Stanford University, began working with gene therapy in 1990. At the time, he recalled, people gave estimates that certain diseases would be cured in five years. .

Kay was part of the team that filed the IND application in 2000, leading to the first attempt to routinely administer AAV2 to humans. The work was done by gene therapy pioneer Katherine High, MD, who was at the University of Pennsylvania at the time. Her (Today, she is a visiting professor at Rockefeller University and professor of therapeutics at AskBio).

Over the years, Kay has learned that AAV doses and transduction efficiencies in animal studies do not necessarily correlate with humans. In short, animal tests make predictions that have nothing to do with humans.

(Video) M. Giacca - AAV-based vectors as powerful tools for gene transfer into post-mitotic..

For example, in previous experiments from 22 years ago, the team found that AAV vectors were not toxic when administered to dogs and rodents. However, humans have elevated liver enzymes. Furthermore, in humans, but not in animals, there is a T cell response against capsids that have been taken up by cells and degraded.

This experience led Kay to find a better model for AAV vector selection. The model is a mouse model containing primary human hepatocytes developed in the laboratory of Markus Grompe, MD, director of the Papé Family Pediatric Research Institute at Oregon Health & Science University. Using this model, Kay's group became the first to use "humanized" mice to compare the transduction efficiency of different AAV vectors.

Kay's group was also the first to use an AAV vector development process in which capsid variants were selected from a capsid library. In this process, scientists disassemble capsids in nature, mix the resulting capsid fragments together, and assemble new capsids at random. Eventually, the capsid becomes part of a capsid library, which may contain millions of capsid variants.

Capsids were injected into humanized mice and capsids successfully transduced into human hepatocytes were recovered. One of the most successful capsids discovered by Kay's group was the AAV-LK03 capsid. It performs 10 times better than AAV2 or AAV8. It is used in LogicBio trials as well as Spark Therapeutics trials. Spark calls the capsid Spark200 and uses it to deliver SPK-8011, a gene therapy for hemophilia A.

Kay screens for AAV vectors with different properties. For example, she examined AAV vectors that can package large amounts of DNA. Detection of AAV vectors that do not elicit an immune response is more difficult, she said. Detecting them is more difficult unless she knows more molecular details about what stimulates these responses.

First up: in vivo genome editing in children

AAV vectors: selection permutations, favorable fates (2)

In 2014, Kay developed work in his lab with two postdocs to form LogicBio. In 2016, LogicBio welcomed a new President and CEO, Fred Chereau, an executive with extensive experience building biotech companies.

In October 2021, LogicBio announced the results of a clinical trial that demonstrated the first use of nuclease-free genome-editing technology in children. The technology was also developed in Kay's lab.

(Video) M. Giacca - AAV-based vectors as powerful tools for gene transfer into post-mitotic cells in vivo

LogicBio is focused on developing gene therapy solutions for pediatric patients with rare diseases. The company's first drug in development (LB-001) is designed to treat methylmalonic acidemia (MMA), a life-threatening condition that affects approximately 1 in 50,000 newborns in the United States.

Early data from a phase I/II trial look promising. Based on the safety data from the first two patients, an independent data safety monitoring committee recommended that the trial continue with the inclusion of younger children and the use of higher doses.

But on February 2, the company held a conference call to reveal that the FDA had suspended the MMA trial. At that time, four patients had already taken the drug. The first two patients, the eldest, are fine. But the third and fourth patients experienced thrombotic microangiopathy (TMA), a previously reported side effect with other AAV gene therapies.

Chereau said both patients are doing well and have returned home. At LogicBio, the researchers are working on implementing changes to get the experiment working again. Chereau noted that although the TMA phenomenon is not well understood, complement activation is known to be involved. While prophylactic administration of steroids and high-dose AAV vector-delivered therapies are now the standard, more is needed to prevent reactions. To do this, the LogicBio researchers can modify the protocol to add a product (a C5 inhibitor) that inhibits complement activation.

AAV vectors: selection permutations, favorable fates (3)

Gene editing and gene therapy are still in their infancy, and companies in the field are still learning, Chereau said. It takes time for the field to learn how to introduce steroids. Now people in the field know that steroids are good, but probably not good enough. So these guys are considering making other changes to their protocols. For example, an IND application filed by Regenxbio for its DMD program describes a protocol in which AAV8-based gene therapy is combined with strategies to reduce complement activation.

Some researchers focus on other solutions. Some do "stealth AAV" or try to engineer epitopes that tag the AAV to the immune system. Kay noted that it's not clear if they work. She advocates the strategy of finding AAVs that can be given in low doses.

As development programs progress, the field of gene therapy is informed and reacts to the good and the bad. Chereau noted that other gene therapy companies have approached LogicBio considering their own IND applications. And LogicBio discovered that its problems were being shared by other companies, and those companies were willing to share information. When it comes to patient safety, the industry is willing to cooperate. “We all learn from each other,” Chereau declared.

(Video) Lunch & Learn: Intro to Viral Vectors

AAV vectors: selection permutations, favorable fates (4)

Gene therapy for inner ear diseases

An ear for music can come with a mind for science. Check out the Longwood Symphony Orchestra (LSO), a group of Boston scientists, doctors, and other health professionals from the Longwood Medical District, home of Harvard Medical School and several hospitals.

The LSO scientist-musician is not alone. Another scientist and musician also active in the Boston area is Akouo's CEO, Dr. Manny Simons. Simmons studied music as an undergraduate at Harvard University. But his interest in neuroscience began to compete for his attention.

In fact, when Simons decided to study biomedical engineering at the Massachusetts Institute of Technology (MIT), the emphasis turned to neuroscience. In the ScD's Robert S. Langer Laboratory, Simons focuses on drug delivery to the inner ear. She received her PhD in 2008 and joined Voyager Therapeutics, an AAV gene therapy company, in 2014. But she never lost her passion for music. For Simmons, this passion has enhanced her appreciation of hearing.

In 2016, Simons co-founded Akouos, which focuses on disorders of the inner ear.Since then, Simmons has been ableCombine an interest in science with a commitment to developing treatments to restore hearing. His company has two outstanding projects. Both can appear on IND applications at the end of the year. If continued, these two programs will be the first two inner ear AAV programs to enter clinical development.

The first program, AK-OTOF, is an AAV gene therapy to restore hearing in patients with hereditary sensorineural hearing loss due to mutations in the otoferlin gene. The otoferlin gene is expressed in sensory cells (hair cells) found in the inner ear and converts sound-driven fluid waves into neural signals. The second program, AK-antiVEGF, is an AAV gene therapy for vestibular schwannoma. AK-antiVEGF is designed to induce cells in the inner ear to secrete antitumor proteins.

To date, no drug or biologic has ever been approved for direct administration into the inner ear. The inner ear is not easily accessible. It is closer to the brain than to the middle ear, with sensory cells encased in a bony, fluid-filled structure.

AAV gene therapy is "very good for the inner ear," Simons says. AAV vectors can be administered directly without exposure to other parts of the body, allowing a high proportion of vectors to reach target cells. Furthermore, hair cells do not divide, which means that the expression of functional genes can continue in the long term. (Because AAV vectors deliver genes as episomes, DNA that doesn't integrate into the cell's genome, cleavage means dilution.)

(Video) Integrating Novel Advances in Gene Delivery and Genome Engineering for Therapeutic Application

Furthermore, AAV vectors were relatively well tolerated in the immunocompromised inner ear. In Akouos's tests with non-human primates, the AAV vectors did not elicit an immune response, even when administered in high doses. Still, Akouos used lower doses of AAV vectors than are used in traditional gene therapy. Normally, the dose of AAV vector delivered into the bloodstream is 1014o 1015virus particles per kilogram. Akouos used doses three to four orders of magnitude lower than that. Together, these factors take many worries out of Akouos' job.

accidental coincidence of factors

When Akouos was founded, Simons and the company's other co-founders, Michael McKenna, MD, and Bill Sewell, MD, focused on the surgical delivery methods required to deliver vectors into the inner ear. But they don't have vectors. In fact, at that time it was not known whether a capsid with tropism for the inner ear existed.

It didn't take long for Akuos to get lucky. He comes from the laboratory of Dr. Luk Vandenberghe, director of the Grousbeck Genn Treatment Center at Massachusetts Eye and Ear. Vandenberghe constructed an ancestral library of AAV capsids using the capsid sequences of all naturally known AAV capsids and computationally predicting ancestry from him. He then developed a synthetic AAV vector that could safely and efficiently transfer the gene into the mammalian inner ear (Landegger et al., 2017).Evening. Biotechnology.2017; 35 (3): 280-284).

Vandenberghe demonstrated that the AAV Anc80 vector has a high transduction efficiency of inner ear hair cells. Akouos licensed the entire library (38,000 different capsids) for all inner ear applications. Combining Vandenberghe's Anc80 with a surgical application method for the inner ear means the company is moving forward. These factors "come together to open up a new field in medicine," Simmons said.

So Simmons is worried about a lawsuit? He admitted that he was the first of his kind and that children were recruited. Until the trial begins, the company won't know if the initial dose will have an effect or if children will benefit.

The obstacles to gene therapy are many and difficult to overcome. Even with fewer immune system complications, such as Akouos's work with the inner ear, the question of efficacy remains.

Reflecting on her early days in the field, Kay says that even small advances are cause for celebration: "If we could turn a cell into a positive mouse, we'd be jumping." Inspiration was found in bone marrow development and transplantation. These patterns took decades to build, proving that patience can be rewarded.

(Video) Capsid titre quantification for AAV based therapeutics

Many different types of methods can help. As Kay said, "We don't know which platform is better." She added that there are right and wrong ways to do things. But there is no real way to go.


What factors must be considered when choosing a vector for gene therapy trial? ›

Important parameters to be considered when choosing a gene therapy vector include: (1) size limitations for insertion of transgenes, (2) purity and titer of the vector, (3) transduction efficiency, (4) ability to infect dividing and/or quiescent cells, (5) long-term expression of transgenes, (6) integration into the ...

What are the limitations of AAV? ›

The primary limitations of AAV vectors are their small cloning capacity and their elicitation of a humoral immune response. AAV vectors exhibit a number of properties that have made this vector system an excellent choice for both CNS gene therapy and basic neurobiological investigations.

Is AAV bsl1 or bsl2? ›

Historically, the IBC has assigned all work with AAV/rAAV to Biosafety Level 2 (BSL-2) or Animal Biosafety Level 2 (ABSL-2).

How long is AAV stable at 4c? ›

However if the virus is repeated freeze-and-thaw, it will cause significant decrease of titer. For short-term storage, AAV is stable at 4? C for up to almost 2-3 weeks.

What are the basic criteria for choosing vector? ›

A cloning vector should possess an origin of replication so that it can self-replicate inside the host cell. It should have a restriction site for the insertion of the target DNA. It should have a selectable marker with an antibiotic resistance gene that facilitates screening of the recombinant organism.

What are the four factors to consider when choosing vectors for recombinant proteins? ›

When you are choosing an expression vector, you'd better keep these things in mind:
  • Expression System. You need to choose an expression vector that is specific for the expression system. ...
  • Purification Tag. ...
  • Applications of the Target Protein. ...
  • Strength of the Promoter. ...
  • Selectable Marker. ...
  • Vector Size.

What are the disadvantages of AAV in gene therapy? ›

As discussed in regards to the Adenoviral vector, transgenes that remain as episomes instead of integrating into the host genome are at the risk of being lost or degraded when that cell undergoes mitotic division. This is not only a disadvantage of using the AAV as the vector it is also a risk to the patient.

What are the disadvantages of viral vectors for gene therapy? ›

The main drawbacks of using virus vectors are its immunogenicity and cytotoxicity. The first related fatality of gene therapy clinical trial was related to the inflammatory reaction to the viral vector (Adenovirus).

Why AAV is it mostly preferred as a viral vector in gene replacement therapies? ›

Adeno-associated virus (AAV) vectors have been frequently used for gene therapy applications due to the lack of pathogenicity and toxicity, ability to infect both dividing and non-dividing cells, and long-term transgene expression profiles. Furthermore, the safety profile in various clinical trials has been good.

What biosafety level is AAV vector? ›

AAV vectors are biosafety level 1, and consist of recombinant transgene sequences (e.g. marker or human genes) flanked by the AAV inverted terminal repeats. The removal of the majority of viral structural genes renders the vector replication-defective and dependent on an AAV helper virus.

How long is AAV stable at room temperature? ›

Due to the high stability of the capsid, AAV can remain infectious for at least a month at room temperature following simple dessication or lyophilization.

What is the maximum size of the therapeutic gene that can be delivered by AAV? ›

Adeno-associated virus (AAV) vector genomes have been limited to 5 kilobases (kb) in length because their packaging limit was thought to be similar to the size of the parent AAV genome.

How do I increase my AAV titer? ›

Combinations of NaCl and KCl or phosphate-buffered saline (PBS) also increase AAV vector titer.

What temperature does AAV denature? ›

All the serotypes were denatured at 95°C. This experiment revealed a clear differential thermal stability of the AAV particles analyzed.

Is AAV transient or stable? ›

Which Type of Virus Should I Use?
VirusExpressionImmune Response
AAVTransient or Stable*Very Low

What are the 3 rules of vectors? ›

rule 1 - There exists a zero vector. rule 2 - A vector A multiplied by a scalar m is a vector, unchanged in direction, but modified in length by the factor m. rule 3 - The negative of a vector is the original vector flipped 180 degrees;.

What 2 characteristics must a vector have? ›

Vector quantities have two characteristics, a magnitude and a direction.

What are the three important features for a vector? ›

The characteristics of cloning vectors:
  • A cloning vector generally refers to a small DNA fragment into which a foreign DNA of interest can be inserted.
  • The vector DNA should be of a small size.
  • It must be self-replicating inside the host cell.
  • The vector DNA must have an ori(locus) and restriction site.

What are the 3 essential components to a vector plasmid used in cloning? ›

A plasmid contains three necessary functional regions: an origin of replication, a polylinker to clone the gene of interest (called multiple cloning site where the restriction enzymes cleave), and an antibiotic resistance gene (selectable marker).

What are the three most common vectors used in gene therapy? ›

Viral vectors: adenoviral, retroviral lentiviral and AAVs

The fundamental goal of any virus is to survive. To do this, viruses introduce genetic material into host cells in order to replicate, making them fantastic resources for gene therapy.

What are the best vectors for gene therapy? ›

The most commonly used viral vectors are adenovirus, AAV, and lentivirus. Viral vectors work well in gene therapy, but also have some risks. It's possible that your immune system reacts to the virus and causes inflammation.

What are the limitations of adenoviral vectors? ›

The major disadvantage is that adenoviral proteins are highly immunogenic and many people have circulating antibodies and cellular immune responses toward the virus. Other drawbacks are that transgene expression is relatively unstable because the virus remains episomal, and that the virus may be toxic at high doses.

Why is AAV a good vector? ›

AAV vectors are promising therapeutic gene carriers because they can transduce nondividing cells, lack viral genes encoding immunogenic proteins, can be prepared at high titers, and achieve long-term transgene expression in animal models.

Why is AAV better than lentivirus? ›

AAVs are smaller particles than LVs, which gives them advantage on their spreading efficiency within particular tissues. But this benefit also brings a major drawback limiting the size of the expression cassette to 4,5 kb max whereas a lentiviral vector can carry a 10kb insert.

What are viral vectors drawbacks? ›

... While viral vectors are extremely effective in gene transfer, they can also cause safety concerns; including, immune reactions to viruses, limitation in packaging capacity and insertion mutagenesis [140] .

What are the challenges of viral vectors? ›

Choosing the right production system, optimizing DSP, and developing standardized CMC methods and quality assays continue to be the most common challenges faced in viral-vector manufacturing.

What are 3 cons of gene therapy? ›

  • Expensive. Gene therapy can be extremely pricey, making it inaccessible for some people. ...
  • Experimental. Gene therapy is relatively new and there's still a lot about it that we don't know. ...
  • Potentially dangerous. ...
  • Ethical issues. ...
  • May cause infection.
Feb 17, 2022

What is the best viral vector for gene therapy? ›

Adeno‐associated virus (AAV) is the most widely used viral vector for in vivo gene therapy applications. AAV is a nonpathogenic parvovirus with a 4.7 kb DNA genome enclosed in a nonenveloped icosahedral capsid. AAV has 11 natural serotypes and over 100 variants.

Which viral vector is most suitable for gene therapy? ›

AAV is one of the most common vectors used in gene therapy. One of the primary reasons for using AAV is that AAV has a long-term and efficient transgene expression in various cell types in many tissues such as liver, muscle, retina, and the central nervous system (CNS).

What are the advantages and disadvantages of using viral vectors for gene therapy? ›

Table 1
Provide greater gene transfer efficiency in both in vivo and in vitro environmentsCan trigger severe immune responses and inflammatory reactions
Persist for longer periods of time in most casesTheir cloning capacity is very limited
4 more rows
Jul 30, 2022

What is the ratio of viral proteins in AAV? ›

Despite serotype diversity, all AAV capsids are comprised of 60 copies of three capsid viral proteins (VP; VP1/VP2/VP3) assembled into an icosahedron at a molar ratio of approximately 1:1:10 (VP1:VP2:VP3).

What are the 4 types of viral vectors? ›

There are four main types of viral vectors (adeno-associated viral, adenoviral, lentiviral, retroviral) each with their own unique characteristics, uses, and limitations.

What plasmid is used for AAV production? ›

You just need three plasmids to start making the AAVs you need for your experiment: the packaging plasmid which contains the AAV structural and packaging genes, the adenoviral helper plasmid which contains the proteins needed for the virus to replicate, and the transfer plasmid which contains the viral genome.

Does AAV go to the nucleus? ›

Studies of AAV biology reveal that AAV accumulates in the perinuclear region of cells, presumably unable traffic into the nucleus. The mechanism by which AAV enters the nucleus is largely unknown.

What is the maximum length of DNA that can be inserted in the adeno associated virus? ›

The amount of DNA that can be effectively packaged into adenovirus virions is ~105% of the wild-type genome and thus allows for the insertion of ~2 kb of foreign DNA. With the E1A-deleted viruses, it has been possible to insert 5 to 6 kb of DNA.

What is the half life of AAV capsid? ›

rAAV1. 9-3 had the AAV1 capsid with a 113-amino acid (a.a.) stretch (a.a 455–568) replaced with a.a. 456–568 of the AAV9 capsid (Figure 2a). This modification of the AAV1 capsid substantially delayed the blood clearance and prolonged its half-life from t1/2c:124 hours = 3.3 to 23.8 hours.

Can AAV infect non dividing cells? ›

Adeno-associated virus (AAV) vectors are able to transduce a wide range of dividing and non-dividing cell types, which has made these vectors an important tool for gene therapy.

What is the package limit for AAV? ›

Adeno-associated virus (AAV) has many features which make it a great viral vector, but its packaging capacity is limited to ~4.7kb, or roughly half the packaging limits of lentiviral and adenoviral vectors.

What factors must be considered when choosing a vector for a gene therapy trial? ›

Important parameters to be considered when choosing a gene therapy vector include: (1) size limitations for insertion of transgenes, (2) purity and titer of the vector, (3) transduction efficiency, (4) ability to infect dividing and/or quiescent cells, (5) long-term expression of transgenes, (6) integration into the ...

What has to be considered when selecting a vector for gene therapy quizlet? ›

What factors must be considered when choosing a vector for a gene therapy trial? You must take into consideration the size of the gene, the possible immune response, the effectiveness, and what cells your targeting.

What are the characteristics of an ideal vector for gene therapy? ›

An ideal Viral Vector is genetically stable, safe to handle, non-toxic for host cells, has a high packaging capacity and transduction efficiency, and does not elicit an immune response. Several Viral Vector types have been used in gene therapy research and clinical studies during the past decades.

What is the importance of selecting an appropriate vector for the gene of interest? ›

Selecting a vector is key to experimental design. Your vector choice is also important for us to know to build your construct quickly and efficiently. Once we synthesize your gene we offer several options for vector delivery designed to provide you with the maximum flexibility and speed of delivery.

Which of the following is considered as the vector for gene therapy? ›

AAV is one of the most common vectors used in gene therapy. One of the primary reasons for using AAV is that AAV has a long-term and efficient transgene expression in various cell types in many tissues such as liver, muscle, retina, and the central nervous system (CNS).

What features must be present in the vector in order to express a human gene in bacteria? ›

An expression vector must have elements necessary for gene expression. These may include a promoter, the correct translation initiation sequence such as a ribosomal binding site and start codon, a termination codon, and a transcription termination sequence.

What will be the suitable vector to be used to deliver the desirable gene in developing transgenic plants? ›

The Ti plasmid vector. In plant genetic engineering, the Ti plasmid can be used to carry foreign genes into plant cells. The Ti plasmid is the disease-causing agent of the soil-borne bacteria Agrobacterium tumefaciens.

Which of the following vector is best for gene transfer? ›

Lamda bacteriophages are used as vectors for delivering gene of interest in bacteria.

What are the four hallmarks of successful gene delivery in gene therapy trials? ›

To be successful, a vector must:
  • TARGET the right cells. If you want to deliver a gene into cells of the liver, it shouldn't wind up in the big toe.
  • INTEGRATE the gene in the cells. ...
  • ACTIVATE the gene. ...
  • AVOID harmful side effects.

What is characteristic of a good vector? ›

Common features of an ideal vector include the following: Presence of origin of replication (ori) Selectable markers. Sites for the action of restriction endonucleases (restriction sites)

Which viral vector is most suitable for gene therapy comment? ›

Adenovirus vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene. Such vectors are used for gene therapy, as vaccines, and for cancer therapy. Replication-competent (oncolytic) vectors are employed for cancer gene therapy.

What are the four properties of vectors? ›

Properties of Vector - Equality, Vector Addition, Subtraction and Multiplication.

What is the most important feature in a plasmid to serve as a vector in gene cloning experiment? ›

So, the correct answer is 'Origin of replication(ori)'

What are the two most commonly used genetic vectors? ›

Plasmids and bacteriophages are the two most used forms of vectors.

What is the most promising vector for human gene therapy? ›

Due to these unique features, AAV is the most suitable viral vector for in vivo gene therapies, especially for conditions that require long‐term gene modifications.

What are the 4 organic vectors of gene therapy? ›

Vectors are essentially vehicles designed to deliver therapeutic genetic material, such as a working gene, directly into a cell. There are four main types of viral vectors (adeno-associated viral, adenoviral, lentiviral, retroviral) each with their own unique characteristics, uses, and limitations.


1. Innovations in viral vector development, scale up and production webinar
(Thermo Fisher Scientific)
2. Macrophage Infection by HIV: Implications for Pathogenesis and Cure: Day Two
(National Institute of Mental Health (NIMH))
3. Accelerating Viral Vector Commercialisation Through a Suspension Platform
4. M. Giacca - Gene therapy of cardiovascular disorders
5. Gene Therapy in Neuroscience 101
(Harvard Brain Science Initiative)
6. Advanced Biomarker Tools to Support Gene Therapy Research


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