Requirements for market entry of gene drive-modified mosquitoes for control of vector-borne diseases: analogies to other biologic and biotechnology products

Gene drive-modified mosquitoes (GDMMs) are proposed as new tools for control and elimination of malaria and other mosquito-borne diseases, and promising results have been observed from testing conducted in containment. Although still at an early stage of development, it is important to begin now to consider approval procedures and market entry strategies for the eventual implementation of GDMMs in the context of disease control programs, as these could impact future research plans. It is expected that, as for other types of new products, those seeking to bring GDMMs to market will be required to provide sufficient information to allow the regulator(s) to determine whether the product is safe and effective for its proposed use. There already has been much emphasis on developing requirements for the biosafety components of the “safe and effective” benchmark, largely concerned with their regulation as genetically modified organisms. Other potential approval requirements have received little attention, however. Although GDMMs are expected to be implemented primarily in the context of public health programs, any regulatory analogies to other public health products, such as pharmaceuticals, vaccines, or chemical pesticides, must take into account the characteristics of live mosquito products. Typical manufacturing standards related to product identity, potency or quality will need to be adapted to GDMMs. Valuable lessons can be drawn from the regulatory approval processes for other whole organism and genetically modified (GM) organism products. Supply chain requirements, such as scale of production, location and design of production facilities, and methods of distribution and delivery, will be dependent upon the characteristics of the particular GDMM product, the conditions of use, and the region to be served. Plans for fulfilling supply chain needs can build upon experience in the development of other live insect products for use in public health and agriculture. Implementation of GDMMs would benefit from additional research on enabling technologies for long-term storage of mosquito life stages, efficient mass production, and area-wide delivery of GDMMs. Early consideration of these practical requirements for market entry will help to mitigate downstream delays in the development of these promising new technologies

Systematic variation in mRNA 3′-processing signals during mouse spermatogenesis

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis

Infertility with Impaired Zona Pellucida Adhesion of Spermatozoa from Mice Lacking TauCstF-641

Fertilization is a multistep process requiring spermatozoa with unique cellular structures and numerous germ cell-specific molecules that function in the various steps. In the highly coordinated process of male germ cell development, RNA splicing and polyadenylation help regulate gene expression to assure formation of functional spermatozoa. Male germ cells express tauCstF-64 (Cstf2t gene product), a paralog of the X-linked CstF-64 protein that supports polyadenylation in most somatic cells. We previously showed that loss of tauCstF-64 causes male infertility because of major defects in mouse spermatogenesis. Surprisingly, although Cstf2t−/− males produce very few recognizable spermatozoa, some of the spermatozoa produced are motile. This led us to ask whether these Cstf2t−/− sperm were fertile. A motile cell-enriched population of spermatozoa from Cstf2t-null males dispersed cumulus cells of cumulus-oocyte complexes normally. However, motile spermatozoa from Cstf2t-null males failed to fertilize cumulus-intact mouse eggs in vitro. In addition, sperm adhesion to the zona pellucida (ZP) of cumulus-free eggs was significantly decreased, indicating tauCstF-64 is required for production of spermatozoa capable of ZP interaction. Acrosomal proteins involved in sperm-ZP recognition, including zonadhesin, proacrosin, SPAM1/PH-20, and ZP3R/sp56, were normally distributed in the apical head of Cstf2t−/− spermatozoa. We conclude that tauCstF-64 is required not only for expression of genes involved in morphological differentiation of spermatids but also for genes having products that function during interaction of motile spermatozoa with eggs. To our knowledge, this is the first demonstration that a gene involved in polyadenylation has a negative consequence on sperm-ZP adhesion

Loss of polyadenylation protein τCstF-64 causes spermatogenic defects and male infertility

Polyadenylation, the process of eukaryotic mRNA 3′ end formation, is essential for gene expression and cell viability. Polyadenylation of male germ cell mRNAs is unusual, exhibiting increased alternative polyadenylation, decreased AAUAAA polyadenylation signal use, and reduced downstream sequence element dependence. CstF-64, the RNA-binding component of the cleavage stimulation factor (CstF), interacts with pre-mRNAs at sequences downstream of the cleavage site. In mammalian testes, meiotic XY-body formation causes suppression of X-linked CstF-64 expression during pachynema. Consequently, an autosomal paralog, τCstF-64 (gene name Cstf2t ), is expressed during meiosis and subsequent haploid differentiation. Here we show that targeted disruption of Cstf2t in mice causes aberrant spermatogenesis, specifically disrupting meiotic and postmeiotic development, resulting in male infertility resembling oligoasthenoteratozoospermia. Furthermore, the Cstf2t mutant phenotype displays variable expressivity such that spermatozoa show a broad range of defects. The overall phenotype is consistent with a requirement for τCstF-64 in spermatogenesis as indicated by the significant changes in expression of thousands of genes in testes of Cstf2t −/− mice as measured by microarray. Our results indicate that, although the infertility in Cstf2t −/− males is due to low sperm count, multiple genes controlling many aspects of germ-cell development depend on τCstF-64 for their normal expression. Finally, these transgenic mice provide a model for the study of polyadenylation in an isolated in vivo system and highlight the role of a growing family of testis-expressed autosomal retroposed variants of X-linked genes

Polyadenylation proteins CstF-64 and τCstF-64 exhibit differential binding affinities for RNA polymers

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, τCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between τCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of τCstF-64 and CstF-64 have different affinities for RNA elements. We quantified K(d) values of CstF-64 and τCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)(18), poly(A)(18) or poly(C)(18), with affinity for poly(C)(18) being the lowest. However, CstF-64 had a higher affinity for poly(U)(18) than τCstF-64, whereas it had a lower affinity for poly(GU)(9). Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)(18), but changes in amino acids downstream of the C-terminal α-helical region decreased affinity towards poly(U)(18). Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is important in RNA sequence recognition. This supports the hypothesis that τCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements