AAV Vector Design Services

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Adeno-associated virus (AAV) vectors have become an indispensable tool in gene therapy. Protheragen's comprehensive AAV vector design services leverage our deep understanding of AAV biology and our extensive toolbox of natural and engineered capsids to deliver tailored solutions for our clients' gene therapy needs.

Introduction to AAV Vector Design

AAV vector design is a cutting-edge scientific discipline that lies at the intersection of molecular biology, genetic engineering, and virology. It involves the manipulation and optimization of AAV vectors for efficient and targeted gene delivery. AAV vectors are small, non-pathogenic viruses that have evolved into powerful tools for gene therapy due to their ability to integrate foreign DNA into a host genome without causing disease. These vectors are engineered to carry therapeutic genes to specific cells or tissues, offering a promising approach for treating a wide range of genetic disorders and diseases.

AAV capsid engineering design strategies. Fig.1 Rational design strategies for AAV capsid engineering. (Lee E. J., et al., 2018)

AAV (Adeno-Associated Virus) vector design is a cutting-edge scientific discipline that lies at the intersection of molecular biology, genetic engineering, and virology. It involves the manipulation and optimization of AAV vectors for efficient and targeted gene delivery. AAV vectors are small, non-pathogenic viruses that have evolved into powerful tools for gene therapy due to their ability to integrate foreign DNA into a host genome without causing disease. These vectors are engineered to carry therapeutic genes to specific cells or tissues, offering a promising approach for treating a wide range of genetic disorders and diseases.

Types of AAV Vector Capsids

AAV vectors are classified into multiple serotypes, each with distinct tissue tropisms and transduction efficiencies. The choice of serotype is critical for the success of gene therapy, as it influences the vector's ability to reach and infect target cells.

AAV1-AAV9: These serotypes have been extensively studied and exhibit different tissue preferences, with some showing affinity for muscles, liver, or neurons.
Engineered Serotypes: Advanced molecular techniques have led to the creation of novel serotypes with improved properties, such as enhanced tissue specificity or reduced immunogenicity.

Table 1. Commonly used AAV vector capsids, their origin, receptor usage, and in vivo tropism. (Colón-Thillet R., et al., 2021)

AAV capsid	Naturally occurring	Engineered	Likely species origin	AAV Receptor usage	Co-receptors	Other cellular receptor	Tissue culture activity	In vivo tissue tropism
AAV1	Yes	No	NHP	Yes	-	Sialic acid	Moderate	Skeletal muscle, CNS, airway, retina, heart, liver
AAV2	Yes	No	Human	Yes	FGFR-1, HGFR, αVβ1 and αVβ5 integrins, Laminin receptor, CD9	HSPG	Good	Skeletal muscle, CNS, retina, liver
AAV3	Yes	No	Human	Yes	FGFR-1, HGFR, Laminin receptor	HSPG	Moderate	Skeletal muscle, liver
AAV4	Yes	No	African green monkey	No	Unknown	Sialic acid	Poor	CNS, retina, kidney, lung
AAV5	Yes	No	Human	Yes	PDGFR	Sialic acid	Poor	Skeletal muscle, CNS, airway, retina
AAV6	Yes	No	Human	Yes	EGFR	HSPG, Sialic acid	Moderate	Skeletal muscle, airway, heart
AAV7	Yes	No	Rhesus macaque	Unknown	Unknown	Unknown	Poor	Skeletal muscle, CNS, retina, liver
AAV8	Yes	No	Rhesus macaque	Yes	Laminin receptor	Unknown	Poor	Skeletal muscle, CNS, airway, retina, heart, liver
AAV9	Yes	No	Human	Yes	Laminin receptor	Galactose	Poor	Skeletal muscle, CNS, airway, retina, heart, liver
AAV.rh10	Yes	No	Rhesus macaque	Unknown	Laminin receptor	Unknown	Poor	Skeletal muscle, CNS, airway, retina, heart, liver
AAV.DJ	No	Yes	NA	Unknown	Unknown	HSPG	Good	Liver, CNS, retina
AAV.LK03	No	Yes	NA	Unknown	Unknown	Unknown	Poor	Human liver

Our Services

Protheragen provides a suite of AAV vector design services tailored to the precise needs of gene therapy research and development. Our services are structured to cover every aspect of AAV vector development, from DNA fragment preparation to final delivery.

Preparation of DNA Fragments

Digestion Fragments from Plasmids

We utilize restriction enzymes to precisely cut plasmids, releasing the desired DNA fragments for subsequent cloning into AAV vectors.

Amplification of Requested Sequences

Employing PCR and other amplification techniques, we ensure that even rare sequences are present in sufficient quantities for vector construction.

Synthesis of DNA Fragments with Low Cost

We offer cost-effective DNA fragment synthesis making large-scale projects feasible without compromising on budget.

Molecular Cloning

Cloning with Standard Cloning Techniques: With standard cloning techniques such as Gibson assembly and Gateway cloning to insert DNA fragments into AAV vectors, ensuring high fidelity and efficiency.
Multiple Fragments Assembled Together: Enabling the simultaneous assembly of multiple DNA fragments, streamlining the creation of complex AAV vectors.

Quality Control

Restriction Enzyme Digestion
This step verifies the presence of correct clones by cutting DNA at specific sites, ensuring the accuracy of AAV vector construction.
SmaI/AdhI Digestion to Confirm ITRs
We confirm the integrity of the AAV vector's Inverted Terminal Repeats (ITRs), which are essential for vector replication and packaging.

Sanger Sequencing to Verify Inserts
Sanger sequencing is performed to validate the insertion of DNA sequences within the AAV vector, ensuring sequence accuracy.
Whole Plasmid Sequencing on Request
For projects requiring comprehensive sequence analysis, we offer whole plasmid sequencing services to provide a complete view of the vector's DNA sequence.

At Protheragen, we understand that every gene therapy project has unique requirements. Whether you require a specific tissue tropism, enhanced immune evasion, or a unique genetic payload, our team will work closely with you to develop a tailored solution that maximizes the therapeutic potential of your gene therapy. If you are interested in our services, please feel free to contact us for more details and quotation information of related services.

References

Lee Esther J., Caitlin M. Guenther, and Junghae Suh. "Adeno-associated virus (AAV) vectors: rational design strategies for capsid engineering." Current opinion in biomedical engineering 7 (2018): 58-63.
Colón-Thillet Rossana, Keith R. Jerome, and Daniel Stone. "Optimization of AAV vectors to target persistent viral reservoirs." Virology Journal 18.1 (2021): 85.

All of our services and products are intended for preclinical research use only and cannot be used to diagnose, treat or manage patients.