E2A, VA RNA I, and L4-22k adenoviral helper genes are sufficient for AAV production in HEK293 cells.

The replication-defective adeno-associated virus (AAV) is extensively utilized as a research tool or vector for gene therapy. The production process of AAV remains intricate, expensive, and mechanistically underexplored. With the aim of enhancing AAV manufacturing efficiencies in mammalian cells, we revisited the questions and optimization surrounding the requirement of the various adenoviral helper genes in enabling AAV production.

Boost and Increased Antibody Breadth Following a Second Dose of PARVAX for SARS-CoV-2 in Mice and Nonhuman Primates.

PARVAX is a genetic vaccine platform based on an adeno-associated vector that has demonstrated to elicit potent, durable, and protective immunity in nonhuman primates (NHPs) after a single dose. Here, we assessed vaccine immunogenicity following a PARVAX prime-boost regimen against SARS-CoV-2. In mice, a low-dose prime followed by a higher-dose boost elicited potent neutralizing antibody responses and distinct cross-reactivity profiles, depending on the antigen used in the booster vaccine.

Correlating physicochemical and biological properties to define critical quality attributes of a rAAV vaccine candidate.

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector's physicochemical properties and biological potency. To this end, we evaluated an rAAV-based coronavirus disease 2019 (COVID-19) vaccine candidate that encodes the Spike antigen (AC3) and is produced by a commercially viable process.

Subretinal gene therapy delays vision loss in a Bardet-Biedl Syndrome type 10 mouse model.

Blindness in Bardet-Biedl syndrome (BBS) is caused by dysfunction and loss of photoreceptor cells in the retina. , mutations of which account for approximately 21% of all BBS cases, encodes a chaperonin protein indispensable for the assembly of the BBSome, a cargo adaptor important for ciliary trafficking. The loss of BBSome function in the eye causes a reduced light sensitivity of photoreceptor cells, photoreceptor ciliary malformation, dysfunctional ciliary trafficking, and photoreceptor cell death.

High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography.

The adeno-associated viral vector (AAV) provides a safe and efficient gene therapy platform with several approved products that have marked therapeutic impact for patients. However, a major bottleneck in the development and commercialization of AAV remains the efficiency, cost, and scalability of AAV production. Chromatographic methods have the potential to allow purification at increased scales and lower cost but often require optimization specific to each serotype.

Ancestral library identifies conserved reprogrammable liver motif on AAV capsid.

Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy.

Gene therapy with a synthetic adeno-associated viral vector improves audiovestibular phenotypes in Pjvk-mutant mice.

Recessive PJVK mutations that cause a deficiency of pejvakin, a protein expressed in both sensory hair cells and first-order neurons of the inner ear, are an important cause of hereditary hearing impairment. Patients with PJVK mutations garner limited benefits from cochlear implantation; thus, alternative biological therapies may be required to address this clinical difficulty. The synthetic adeno-associated viral vector Anc80L65, with its wide tropism and high transduction efficiency in various inner ear cells, may provide a solution.

Immunogenicity of an AAV-Based COVID-19 Vaccine in Murine Models of Obesity and Aging.

The SARS-CoV-2 pandemic has had a disastrous impact on global health. Although some vaccine candidates have been effective in combating SARS-CoV-2, logistical, economical, and sociological aspects still limit vaccine access globally. Recently, we reported on two room-temperature stable AAV-based COVID-19 vaccines that induced potent and protective immunogenicity following a single injection in murine and primate models.

Computed tomography and [F]-FDG PET imaging provide additional readouts for COVID-19 pathogenesis and therapies evaluation in non-human primates.

Non-human primates (NHPs) are particularly relevant as preclinical models for SARS-CoV-2 infection and nuclear imaging may represent a valuable tool for monitoring infection in this species. We investigated the benefit of computed X-ray tomography (CT) and [F]-FDG positron emission tomography (PET) to monitor the early phase of the disease in a large cohort (n = 76) of SARS-CoV-2 infected macaques. Following infection, animals showed mild COVID-19 symptoms including typical lung lesions.

Choice of vector and surgical approach enables efficient cochlear gene transfer in nonhuman primate.

Inner ear gene therapy using adeno-associated viral vectors (AAV) promises to alleviate hearing and balance disorders. We previously established the benefits of Anc80L65 in targeting inner and outer hair cells in newborn mice. To accelerate translation to humans, we now report the feasibility and efficiency of the surgical approach and vector delivery in a nonhuman primate model.

Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes.

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment.

Efficient Delivery of Adeno-Associated Virus into Inner Ear Through Trans-Stapes Route in Adult Guinea Pig.

Adeno-associated virus (AAV) are potent vectors to achieve treatment against hearing loss resulting from genetic defects. However, the effects of delivery routes and the corresponding transduction efficiencies for clinical applications remain elusive. In this study, we screened AAV vectors of three serotypes (AAV 8 and 9 and Anc80L65) into the inner ears of adult normal guinea pigs through trans-stapes (oval window) and trans-round window delivery routes .

Efficient cardiac gene transfer and early-onset expression of a synthetic adeno-associated viral vector, Anc80L65, after intramyocardial administration.

Objective: Gene therapy is a promising approach in the treatment of cardiovascular diseases. Preclinical and clinical studies have demonstrated that adeno-associated viral vectors are the most attractive vehicles for gene transfer. However, preexisting immunity, delayed gene expression, and postinfection immune response limit the success of this technology.

ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia.

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose.

An AAV-based, room-temperature-stable, single-dose COVID-19 vaccine provides durable immunogenicity and protection in non-human primates.

The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques.

Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates.

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine.

Homologous Recombination Offers Advantages over Transposition-Based Systems to Generate Recombinant Baculovirus for Adeno-Associated Viral Vector Production.

Viral vectors have a great potential for gene delivery, but manufacturing is a big challenge for the industry. The baculovirus-insect cell is one of the most scalable platforms to produce recombinant adeno-associated virus (rAAV) vectors. The standard procedure to generate recombinant baculovirus is based on Tn7 transposition which is time-consuming and suffers technical constraints.

Gene Therapy Preserves Retinal Structure and Function in a Mouse Model of -Associated Retinal Degeneration.

No treatment is available for nicotinamide mononucleotide adenylyltransferase 1 ()-associated retinal degeneration, an inherited disease that leads to severe vision loss early in life. Although the causative gene, , plays an essential role in nuclear nicotinamide adenine dinucleotide (NAD) metabolism in tissues throughout the body, -associated disease is isolated to the retina. Since this condition is recessive, supplementing the retina with a normal copy of should protect vulnerable cells from disease progression.

Efficient in Utero Gene Transfer to the Mammalian Inner Ears by the Synthetic Adeno-Associated Viral Vector Anc80L65.

Sensorineural hearing loss is one of the most common sensory disorders worldwide. Recent advances in vector design have paved the way for investigations into the use of adeno-associated vectors (AAVs) for hearing disorder gene therapy. Numerous AAV serotypes have been discovered to be applicable to inner ears, constituting a key advance for gene therapy for sensorineural hearing loss, where transduction efficiency of AAV in inner ear cells is critical for success.

Hair Cell Transduction Efficiency of Single- and Dual-AAV Serotypes in Adult Murine Cochleae.

Gene delivery is a key component for the treatment of genetic hearing loss. To date, a myriad of adeno-associated virus (AAV) serotypes and surgical approaches have been employed to deliver transgenes to cochlear hair cells, but the efficacy of dual transduction remains unclear. Herein, we investigated cellular tropism of single injections of AAV serotype 1 (AAV1), AAV2, AAV8, AAV9, and Anc80L65, and quantitated dual-vector co-transduction rates following co-injection of AAV2 and AAV9 vectors in adult murine cochlea.

Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries.

Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members.