Biology subjects

Agbandje-McKenna, M.

Publications and source records attributed to Agbandje-McKenna, M..

6 recordsLinked to original sources

bioRxiv2021

Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ~2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. ImportanceA major byproduct of all Adeno-associated virus (AAV) vector production systems are "empty" capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

molecular biology↗

bioRxiv2020

AAV Capsid Chimeras with Enhanced Infectivity reveal a core element in the AAV Genome critical for both Cell Transduction and Capsid Assembly

Adeno-associated viruses (AAV) have attracted significant attention in the field of gene and cell therapy due to highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV vectors compromised of unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking a novel AAV capable of enhanced transduction of human T cells for applications in immunotherapy, we have developed a unique capsid variant termed AAV X-Vivo (AAV-XV) that is a chimera of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. This AAV chimera showed enhanced infection of human primary T cells and hematopoietic stem cells, and superiority over wildtype AAV6 for the genomic integration of DNA sequences either by AAV alone or in combination with CRISPR gene editing. AAV-XV demonstrated transduction efficiency equivalent to AAV6 at multiplicities of infection 2 logs lower, enabling T cell engineering at low AAV doses. Analyzing the protein coding sequence of AAV-XV revealed disruptions within the assembly-activating protein (AAP) which likely accounted for observed lower virus yield. A series of genome alterations reverting the AAP sequence back to wildtype had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show that AAV-XV is highly efficient at T cell engineering at low AAV dose and demonstrates the importance of AAP coding region in both viral particle assembly and cell infection. IMPORTANCEA major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat-dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy. We have generated a new AAV variant possessing a unique combination of capsid proteins for ex-vivo application termed AAV-XV, which delivers high levels of cell transduction and gene delivery at a lower MOI. Furthermore, we demonstrate a novel finding, and an important consideration for recombinant AAV design, that a region of the AAV genome encoding the capsid protein and AAP gene is critical for both virus yield and the enhancement of infection/transduction.

cell biology↗

bioRxiv2020

Adeno-associated virus capsid assembly is divergent and stochastic

Adeno-associated viruses (AAVs) are small, non-enveloped, and have a T=1 icosahedral capsid. They belong to the Parvoviridae, genus Dependoparvovirus. Interest in AAVs has grown over recent years as they have emerged as promising gene therapy vectors. The AAV capsid, encapsulating the transgene, consists, in total, of 60 subunits made up from three distinct viral proteins (VPs) originating from the same cap gene (VP1, VP2, and VP3), which vary only in their N-terminus. While all three VPs play a crucial and specific role in cell-entry and transduction, their exact stoichiometry and organization in AAV capsids has, despite the availability of several high-resolution structures remained elusive. Here we obtained a set of native mass spectra of intact AAV capsids (Mw {approx} 3.8 MDa) that display both highly resolved regions and regions wherein interferences occur. Through spectrum simulation we resolved and elucidated this spectral complexity, allowing us to directly assess the VP stoichiometries in a panel of serotypes from different production platforms. The data reveals an extremely heterogeneous population of capsids of variable composition. The relative abundance for each of the hundreds of co-occurring capsid compositions is accurately described by a model based upon stochastic assembly from a mixed pool of expressed VP1, VP2, and VP3. We show that even the single-most abundant VP stoichiometry represents only a few percent of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition and arrangement, i.e. no particle is identical. The systematic scoring of the stochastic assembly model against experimental high-resolution native MS data offers a sensitive and accurate new method to characterize these exceptionally heterogeneous gene-delivery vectors.

biochemistry↗

bioRxiv2019

Resolving heterogeneous high-mass macromolecular machineries by Orbitrap-based single particle charge detection mass spectrometry

Here we show that single particle charge-detection mass spectrometry (CD-MS) can be performed on a ubiquitous Orbitrap mass analyser and applied to the analysis of high-mass (megadalton) heterogeneous biomolecular assemblies. We demonstrate that single particle high-mass ions can survive in the Orbitrap for seconds, whereby their measured signal amplitudes scale linearly with charge state over the entire m/z range. Orbitrap based single particle CD-MS can be used to resolve mixed ion populations, accurately predict charge states, and consequently also the mass of the ions. We successfully applied CD-MS to challenging natural and biotherapeutic protein assemblies, such as IgM oligomers, designed protein nano-cages, ribosome particles and intact, empty- and genome-loaded Adeno-associated virus particles. Single particle CD-MS combined with native MS on existing Orbitrap platforms will greatly expand its application, especially in the mass analysis of megadalton heterogeneous biomolecular assemblies.

biophysics↗

bioRxiv2019

An ancient lineage of highly divergent parvoviruses infects both vertebrate and invertebrate hosts.

Chapparvoviruses are a highly divergent group of parvoviruses (family Parvoviridae) first identified in 2013. Interest in these poorly characterized viruses has been raised by recent studies indicating that they are the cause of chronic kidney disease that arises spontaneously in laboratory mice. In this study, we investigate the biological and evolutionary characteristics of chapparvoviruses via comparative analysis of genome sequence data. Our analysis, which incorporates sequences derived from endogenous viral elements (EVEs) as well as exogenous viruses, reveals that chapparvoviruses are an ancient lineage within the family Parvoviridae, clustering separately from members of both currently established parvoviral subfamilies. Consistent with this, they exhibit a number of characteristic genomic and structural features, i.e. a large number of putative auxiliary protein-encoding genes, capsid protein genes non-homologous to any hitherto parvoviral cap, as well as a putative capsid structure lacking the canonical fifth strand of the ABIDG sheet comprising the luminal side of the jelly roll. Our findings demonstrate that the chapparvovirus lineage infects an exceptionally broad range of host species, including both vertebrates and invertebrates. Furthermore, we observe that chapparvoviruses found in fish are more closely related to those from invertebrates than they are to those that infect amniote vertebrates. This suggests that transmission between distantly related host species may have occurred in the past. Our study provides the first integrated overview of the chapparvovirus group, and revises current views of parvovirus evolution\n\nAUTHOR SUMMARYChapparvoviruses are a recently identified group of viruses about which relatively little is known. However, recent studies have shown that these viruses cause disease in laboratory mice and are prevalent in the fecal virome of pigs and poultry, raising interest in their potential impact as pathogens, and utility as experimental tools. We examined the genomes of chapparvoviruses and endogenous viral elements ( fossilized virus sequences derived from ancestral viruses) using a variety of bioinformatics-based approaches. We show that the chapparvoviruses have an ancient origin and are evolutionarily distinct from all other related viruses. Accordingly, their genomes and virions exhibit a range of distinct characteristic features. We examine the distribution of these features in the light of chapparvovirus evolutionary history (which we can also infer from genomic data), revealing new insights into chapparvovirus biology.

evolutionary biology↗

bioRxiv2018

Sub-2 A Ewald Curvature Corrected Single-Particle Cryo-EM

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we have exploited several technical solutions to improve resolution, including sub-Angstrom pixelation, per-particle CTF refinement, and most notably a correction for Ewald sphere curvature. The application of these methods on micrographs recorded on a base model Titan Krios enabled structure determination at [~]1.86-[A] resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle.

biophysics↗