Comparative analysis of GMO regulatory requirements for AAV vectors in the EU and Japan focusing on the shedding data and containment measures.

INTRODUCTION: Recombinant viral-based gene therapy products, such as those incorporating adeno-associated viruses (AAVs), fall under the category of genetically modified organisms (GMOs). The European Union (EU) countries and Japan must obtain environmental risk assessment (ERA) approval for the use of GMOs before starting any clinical trials. It has been reported that the development of GMO-containing products in these two regions encounters several regulatory obstacles due to the longer regulatory procedures and document preparation for ERA. AREAS COVERED: In this article, we comparatively analyzed the ERA document requirements in the EU and Japan for AAV-based recombinant medicinal products to highlight the differences in the context of potential future attempts of convergence. Additionally, we analyzed non-clinical and clinical shedding data requirements, which are key components of ERA reviews in the EU and Japan. Lastly, we compared the containment measures to minimize the spread of GMOs in the environment in the EU and Japan. EXPERT OPINION: Based on our comparative analysis, we present several policy recommendations of standardizing and simplifying the application materials and procedures for the ERA regulations on GMOs in the EU and Japan in the mid-, and long-term timeframe to achieve global regulatory convergence.

Assessing the environmental fate of rAAV in activated sludge and water: Implications for environmental risk assessments and GMO regulatory frameworks.

During the past +20 years, recombinant adeno-associated virus (rAAV) vectors have emerged as the primary vehicle of choice for in vivo gene therapy. rAAV vectors are classified as genetically modified organisms (GMOs), therefore specific biosafety laws apply regarding their use. Environmental agencies participating in the review of clinical trial applications involving viral-based gene therapies (eg based on AAV) focus among other phenomena especially on shedding, a mechanism by which rAAV vectors exit a patient's body and enter the natural environment. For example, following patient use, shed viral particles excreted in urine and feces enter the wastewater treatment facility (WWTF) and subsequently may be released into the natural environment through wastewater effluent discharges. Based on basic molecular biology, it is generally assumed by the scientific community that shed rAAV particles will undergo degradation during the wastewater treatment process. However, despite their importance and increase in use during the last few decades, actual data to support our understanding of the environmental fate of shed rAAV vector particles is unavailable. Data to support this assumption would greatly enhance our knowledge and understanding of degradation kinetics associated with rAAV in the environment. Such data would also provide strong scientific support for changes in current legislation regarding the medicinal use of GMOs. Therefore, the goal of this research was to conduct laboratory experiments to assess the actual environmental fate of rAAV virions. In this study the stability of 4 different rAAV vectors (based on wildtype (wt) AAV serotypes 2,3,6,9) was assessed during incubation in activated sludge (containing live microorganisms). This setting corresponds to conditions as encountered in WWTFs, and has been used in order to assess rAAV fate under environmentally relevant conditions, to gain a better understanding of the general environmental risk posed by shed rAAV particles. The amount of detectable virions in the supernatant, as measured by sensitive and specific qPCR, rapidly decreased within hours and continued to decline, reaching the lower limit of quantitation prior to or by study termination on day 7. Furthermore, a half-life of approximately 7 days for rAAV virions was determined under abiotic conditions, during a room temperature incubation experiment of rAAV vectors in water in the absence of any microbiota or sludge. The findings from this study provide the first insight of its kind into the actual environmental fate of shed rAAV particles, and help the community to better understand the potential impact of rAAVs on the environment. It has become evident now that shed particles are not equipped to remain stable and/or soluble once entering a typical WWTF and therefore do not pose a threat to the natural environment. These findings support a data-driven approach towards a simplified, risk-based regulation of medicinal GMOs in the EU and other regions.

Impact of genetically modified organism requirements on gene therapy development in the EU, Japan, and the US.

Advanced therapies are emerging as an important class of medicinal products; among these, gene therapies are advancing at an exceptional rate. However, one of the major challenges for gene therapies relates to the additional regulatory requirements for genetically modified organisms. In this paper, we provide an overview of the regulatory requirements for genetically modified organisms in the European Union, Japan, and the United States. We share our experience in managing these requirements and their impact on the adeno-associated virus gene therapies that are under development at Pfizer. Specifically, we discuss the relative complexity of the approval process and the impact of risk assessment expectations on the clinical development of genetically modified organisms. We also compare the regulatory processes and timelines of various regions based on our experience with adeno-associated viral vectors. Finally, we propose that genetically modified organisms, for which pathogenicity and replication competency are well controlled, should be regulated solely under medicinal product regulations and be exempt from additional requirements for genetically modified organisms. Even if an exemption is not implemented, it should still be possible to significantly reduce the sponsor and agency burden by simplifying and harmonizing documentation and data requirements as well as timelines for applications for genetically modified organisms.

The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.

Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.

Synthetic lethality in the tobacco plastid ribosome and its rescue at elevated growth temperatures.

Consistent with their origin from cyanobacteria, plastids (chloroplasts) perform protein biosynthesis on bacterial-type 70S ribosomes. The plastid genomes of seed plants contain a conserved set of ribosomal protein genes. Three of these have proven to be nonessential for translation and, thus, for cellular viability: rps15, rpl33, and rpl36. To help define the minimum ribosome, here, we examined whether more than one of these nonessential plastid ribosomal proteins can be removed from the 70S ribosome. To that end, we constructed all possible double knockouts for the S15, L33, and L36 ribosomal proteins by stable transformation of the tobacco (Nicotiana tabacum) plastid genome. We find that, although S15 and L33 function in different ribosomal particles (30S and 50S, respectively), their combined deletion from the plastid genome results in synthetic lethality under autotrophic conditions. Interestingly, the lethality can be overcome by growth under elevated temperatures due to an improved efficiency of plastid ribosome biogenesis. Our results reveal functional interactions between protein and RNA components of the 70S ribosome and uncover the interdependence of the biogenesis of the two ribosomal subunits. In addition, our findings suggest that defining a minimal set of plastid genes may prove more complex than generally believed.

The contributions of wobbling and superwobbling to the reading of the genetic code.

Reduced bacterial genomes and most genomes of cell organelles (chloroplasts and mitochondria) do not encode the full set of 32 tRNA species required to read all triplets of the genetic code according to the conventional wobble rules. Superwobbling, in which a single tRNA species that contains a uridine in the wobble position of the anticodon reads an entire four-fold degenerate codon box, has been suggested as a possible mechanism for how tRNA sets can be reduced. However, the general feasibility of superwobbling and its efficiency in the various codon boxes have remained unknown. Here we report a complete experimental assessment of the decoding rules in a typical prokaryotic genetic system, the plastid genome. By constructing a large set of transplastomic knock-out mutants for pairs of isoaccepting tRNA species, we show that superwobbling occurs in all codon boxes where it is theoretically possible. Phenotypic characterization of the transplastomic mutant plants revealed that the efficiency of superwobbling varies in a codon box-dependent manner, but--contrary to previous suggestions--it is independent of the number of hydrogen bonds engaged in codon-anticodon interaction. Finally, our data provide experimental evidence of the minimum tRNA set comprising 25 tRNA species, a number lower than previously suggested. Our results demonstrate that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA species utilizing standard base pairing or wobbling and by a second tRNA species employing superwobbling. This has important implications for the interpretation of the genetic code and will aid the construction of synthetic genomes with a minimum-size translational apparatus.

Evolutionary constraints on the plastid tRNA set decoding methionine and isoleucine.

The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons.

Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution.

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.