Donald L. Jarvis - US grants

The insect cell-baculovirus expression vector (BEV) system is widely used to produce recombinant proteins and has greatly facilitated basic biomedical research on protein structure, function, and the roles of various proteins in disease. This system also is used to produce recombinant proteins for direct biomedical applications. The use of this system, which will have a profound impact on medicine, ultimately depends upon a clear understanding of protein biosynthesis and processing pathways in insect cells. However, because insect cell protein processing pathways are not a major subject of basic research, important questions remain to be resolved. This proposal focuses on one of these questions: What is the nature of the N-glycosylation pathway in insect cells? Our current view of this pathway is confused and it is difficult to predict what kind of glycan one will find on a recombinant glycoprotein produced in the insect cell-BEV system. We have developed a working model of the insect cell N-glycosylating pathway and the overall goal of this proposal is to evaluate and extend this model. The specific aims are: (1) to introduce additional mammalian processing enzymes into insect cells and determine how this affects their N-glycosylation pathway; (2) to evaluate the capabilities and biomedical applications of insect cell-BEV expression systems with genetically engineered N-glycosylation pathways; (3) to isolate and characterize new insect cell genes encoding glycoprotein processing enzymes; (4) to examine the cell biology and biochemistry of insect cell glycoprotein processing enzymes; (5) to examine the effects of baculovirus infection on the insect cell secretory pathway; and, (6) to determine if insects have beta1, 4-galactosyltransferase. This work has practical value and could yield improved systems for the production of therapeutic glycoproteins or for liver-biosynthetic pathway that clearly differs from the corresponding pathway in higher eucaryotes. A better understanding of the insect cell pathway might help us to understand how protein glycosylation pathways evolved and how carbohydrate processing impacts glycoprotein function. Furthermore, well-defined differences in insect protein glycosylation pathways might be exploited as specific targets for novel pesticide development in the future. This could lead to more effective ways to control medically and agriculturally significant insects and/or the diseases they help to spread, which would have a major impact on worldwide public health.

9818001
Jarvis
The inability of insect cells to generate glycoproteins similar in carbohydrate composition to those of mammalian cells has limited the wider application of insect cells as vehicles for the production of glycoproteins of mammalian origin. While mammalian cells often produce glycoproteins containing complex oligosaccharides terminating in sialic acid, insect cells typically generate glycoforms containing short truncated oligosaccharides terminating in mannose or N-acetylglucosamine. The composition of these covalently-attached carbohydrates, especially the presence of sialic acid, can significantly affect a glycoprotein's structure, stability, biologicaal activity, and in vivo circulatory half-life. Consequently, the objective of this project is to manipulate carbohydrate processing pathways in insect cells in such a way that these cells will produce sialylated glycoproteins. This inter-disciplinary and inter-agency project will involve participating scientists and engineers from Johns Hopkins University, the University of Wyoming, the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and the Forest Service of the Department of Agriculture (USDA).

9814157 Jarvis . The overall goal of these collaborative proposals between the Principal Investigators (PIs) at Iowa and Wyoming is to develop insect cell culture as an alternative to mammalian cell culture by creating a more mammalian glycosylation pattern on recombinant proteins. This will be accomplished in the three main aims of the proposal: Spodoptera frugiperda Sf-9 cells will be altered with genes for galactosyl transferase and sialyl transferase and the effect on the model protein SEAP (secreted alkaline phosphatase) determined. The effect of bioreactor environment on the SEAP glycosylation will be determined, especially carbon dioxide and dissolved oxygen concentrations. Similar studies will be conducted on a much more complex protein model, C1INH, which normally contains six N-linked and seven O-linked glycosylation sites. ***

DESCRIPTION (provided by the applicant): Insect cells are widely known as hosts for baculovirus expression vectors (BEVs), which can produce large amounts of recombinant proteins with eukaryotic modifications. The insect cell-BEV system has been used to produce many different recombinant proteins, facilitating biomedical research on their structures, functions, and roles in disease. Many companies are using the insect cell-BEV system to produce recombinant proteins for high-throughput functional screens and to develop vaccines, therapeutics, and diagnostic reagents. The advantages of using insect cells for these applications include ease of scale-up, lower costs, and the absence of human adventitious agents. These important research activities require a dear understanding of protein biosynthesis and processing pathways in insect cells. However, this has not been a major topic of research. This proposal focuses on the insect cell protein N-glycosylation pathway. The long-term goals are to provide an unequivocal and detailed view of this important metabolic pathway in insect cells and to use this knowledge to develop new insect-based protein production systems with more extensive recombinant glycoprotein processing capabilities. The widespread use of insect cells as protein production systems for many important biomedical research projects is a strong justification for this research. Intelligent use of insect cells for recombinant glycoprotein production will require a better understanding of their N-glycosylation pathway. This understanding can direct innovative metabolic engineering efforts to produce new insect ceft-BEV systems with improved N-glycoprotein processing capabilities. Insect cells also occupy an important niche as models for fundamental research in the emerging field of glycobiology. The insect cell N-glycosylation pathway lies in-between the pathways of lower and higher eukaryotes in complexity. Thus, well-defined differences in insect protein glycosylation pathways could be exploited as specific targets to develop future insecticides. This could lead to better ways to control agriculturally and medically important insects and the diseases they help to spread, which would have a major impact on public health, worldwide.

DESCRIPTION (provided by applicant): Many biomedically significant proteins, including antibodies, cytokines, anticoagulants, blood clotting factors, and others are glycoproteins. Thus, there is a high demand for systems that can be used to produce recombinant glycoproteins for basic biomedical research and direct clinical applications. However, currently available recombinant protein production systems cannot meet this demand. In fact, no currently available system can produce large amounts of recombinant glycoproteins in properly glycosylated form at relatively low cost. The long-term objective of this proposal is to genetically engineer the silkworm to fulfill these requirements and provide a new system for recombinant glycoprotein production. Recent studies have shown that the silkworm silk gland, which is a highly efficient silk protein production and secretion organ, can be genetically engineered to efficiently produce and secrete recombinant proteins. But, transgenic silkworms have been neither developed nor used for recombinant glycoprotein production. The major impediment is that the endogenous protein glycosylation pathways of the silk gland cannot be expected to properly glycosylate higher eukaryotic glycoproteins. We will use metabolic engineering to overcome this impediment, as part of a broader effort to develop the silkworm as a new system for recombinant human glycoprotein production. The basic approach will be to use the piggyBac vector system to isolate transgenic silkworms encoding (1) higher eukaryotic enzymes needed to humanize the native silk gland protein N-glycosylation pathway and (2) a recombinant human N-glycoprotein. Each transgene will be placed under the control of a tissue-specific promoter that will target its expression to the silk gland. There are no previous reports of recombinant glycoprotein production using any type of transgenic insect as a bioreactor. In addition, there are no previous reports of engineering a protein glycosylation pathway in any multicellular animal. Therefore, the proposal to use the silk gland of a transgenic silkworm as a bioreactor for recombinant glycoprotein production and secretion, coupled with the proposal to metabolically engineer the protein N-glycosylation pathway in a major organ of this lower eukaryote, is truly original and innovative. The specific aims of this proposal are (1) To construct and test piggyBac vectors for silk gland-specific expression of genes encoding (1a) enzymes needed to humanize the silkworm protein N-glycosylation pathway and (1b) genes encoding a recombinant human glycoprotein of interest;(2) To use the piggyBac vectors from aim 1 to produce transgenic silkworms;and (3) To assess recombinant glycoprotein production, secretion, and glycosylation by the transgenic silkworms from aim 2.The glycoproteins are a major subclass of proteins distinguished by the presence of carbohydrate side chains covalently linked to the polypeptide backbone. Many different types of biomedically significant proteins, such as antibodies, cytokines, anticoagulants, blood clotting factors are glycoproteins. Modern biomedical researchers studying human glycoproteins or producing them for clinical use rely heavily on recombinant protein production systems. Thus, there is a high demand for systems that can be used to produce recombinant glycoproteins. Unfortunately, few of the currently available systems are well suited for the production of recombinant glycoproteins, as few can produce higher eukaryotic glycoproteins with authentic carbohydrate side chains. Thus, the basic purpose of the research proposed herein is to create a new system that can be used to produce recombinant glycoproteins for basic biomedical research and direct clinical applications. More specifically, we will genetically engineer the silkworm to create this new system. While it might seem strange to target a caterpillar, such as the silkworm, to develop a recombinant glycoprotein production system, we have good reasons to do so. One major reason is that the silkworm silk gland has evolved over millions of years as a highly efficient protein production and secretion organ. Furthermore, several published studies have shown that this organ can be engineered to efficiently produce and secrete recombinant proteins. However, silkworms have not been used for recombinant glycoprotein production because their endogenous protein glycosylation pathways cannot properly glycosylate foreign, higher eukaryotic glycoproteins. Together, the Jarvis and Fraser labs are uniquely positioned to address this problem. The Jarvis lab has been studying and engineering insect protein glycosylation pathways for the past decade and the Fraser lab has developed a superb system for efficient genetic transformation of insects, particularly the silkworm. Thus, we plan to combine our skills to isolate transgenic silkworms that will encode both the higher eukaryotic enzymes needed to humanize their protein glycosylation pathway and a biomedically relevant human glycoprotein of interest. Importantly, the expression of each transgene will be specifically targeted to the silk gland placed using a tissue-specific promoter. There are no previous reports of recombinant glycoprotein production using any type of transgenic insect as a bioreactor. There also are no previous reports of engineering a protein glycosylation pathway in any multicellular animal. Therefore, our proposal to use the silk gland of a transgenic silkworm as a bioreactor for recombinant glycoprotein production and secretion, coupled with our proposal to metabolically engineer the protein N-glycosylation pathway in a major organ of this lower eukaryote, is truly original and innovative. The successful development of the silkworm as a system for recombinant glycoprotein production would have a broad impact with implications in many areas of biomedical research. A better tool for recombinant glycoprotein production would facilitate basic research on glycoprotein structure and function. It also could be used in the biotechnology industry to produce recombinant glycoproteins for clinical use as vaccines or therapeutics. Again, while it might seem like a strange platform, the idea to use caterpillars for the production of non-glycosylated proteins has already been commercialized (see www.c-perl.com). The biotechnological impact of this system could be huge, considering that many high profile, clinically relevant proteins, such as antibodies (e.g. herceptin.), cytokines (e.g. EPOGEN), and anticoagulants (e.g. Tenecteplase") are glycoproteins. At a more basic level, the metabolic engineering effort, which is key to this project, represents an elaborate ectopic expression experiment that will broadly address the biological significance of the differences in protein N-glycosylation pathways of lower and higher eukaryotes. These results will be of great interest to basic scientists, particularly glycobiologists studying protein N-glycosylation in lower organisms and the evolution of protein glycosylation pathways. Finally, these results will be of great interest to bioengineers working to overcome the evolutionary limitations of lower eukaryotic systems for recombinant glycoprotein production.

[unreadable] DESCRIPTION (provided by applicant): Silk fibers have many current and future biomedical applications. They are widely used as fine suture materials and even thinner fibers are needed for ocular, neurological, and cosmetic surgeries. Silk fibers also hold promise as materials for artificial ligaments and tendons and they have many other potential biomedical applications. Many different recombinant protein production systems have been used to try to meet current needs and to develop additional biomedical applications of silks. Each has yielded silk proteins, but none has consistently yielded useful silk fibers. Thus, the overall purpose of this R21 exploratory proposal is to develop a system that can produce spider silk fibers. Our basic approach will be to adopt the silkworm as a surrogate host for spider silk protein production. The highly efficient piggybac system will be used to genetically transform mutant silkworms, which produce no native silk, with a synthetic gene encoding an unusually large spider silk protein. The genetic and biochemical properties of the resulting transgenic silkworms, particularly their ability to produce spider silk fibers, will then be critically assessed. A key feature of our plan is that it includes specific measures that will optimize our ability to obtain fibers. These include: (i) using a synthetic gene encoding an unusually large spider silk protein, (ii) using a promoter that targets expression of the heterologous silk proteins to the silk gland, which is naturally equipped to spin silk fibers, and (iii) using an appropriate leader peptide for secretion from the silk gland, and (iv) using a surrogate host, the silk moth, which is highly amenable to genetic transformation and naturally equipped to spin silk fibers. The successful isolation of a transgenic silkmoth that can produce spider silk fibers in this exploratory project will set the stage for future projects designed to further develop this system to produce spider silk fibers with pre-determined physical properties optimized for specific biomedical applications. This will exploit current knowledge of specific peptide motifs contributing tensile strength or elasticity to spider silks. Theoretically, these motifs can be combined in various ways to design silk fibers differing in strength and elasticity. The overall likelihood that the current project can be successfully completed is enhanced by the fact that it will be undertaken by a team of three researchers with established, complementary programs in three areas key to the project: spider silks (Lewis), insect expression systems (Jarvis), and insect transformation (Fraser). Silk fibers have many current and future biomedical applications. They are widely used as fine suture materials and even thinner fibers are needed for ocular, neurological, and cosmetic surgeries. Silk fibers also hold promise as materials for artificial ligaments and tendons and they have many other potential biomedical applications. Many different recombinant protein production systems have been used to try to meet current needs and to develop additional biomedical applications of silks. Each has yielded silk proteins, but none has consistently yielded useful silk fibers. Thus, the overall purpose of this R21 exploratory proposal is to develop a system that can produce spider silk fibers for these current and future biomedical applications. Our plan for this exploratory project is to isolate a transgenic silkworm that can produce spider silk fibers. If this project is successful, it will set the stage for future projects designed to further develop this system to produce spider silk fibers with pre-determined physical properties optimized for specific biomedical applications. These projects will exploit current knowledge of specific peptide motifs contributing tensile strength or elasticity to spider silks. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Elucidating the cellular mechanisms of prion propagation and clearance for devising new targets for intervention in prion disease There are an increasing number of neurodegenerative disorders which result from the aggregation of misfolded proteins and which share patho-physiological mechanisms. Prion diseases are the prototypical protein misfolding diseases, and their pathogenesis is associated solely with aberrant misfolding of a single cellular protein (PrPc). Prion diseases are unique in this group as they are infectious disorders found in man and animals. Besides sporadic or genetic manifestation, they can be acquired by infection and transmitted between species, resulting in endemic or epidemic scenarios (e.g. BSE/vCJD and CWD). They can be controlled, but eradication is impossible. Therefore, it is mandatory to understand the molecular and cellular requirements for propagation and transmission of prions in order to device rational strategies for controlling these events. Advances in understanding prion patho-physiology will have major implications for other protein misfolding diseases, as it may help elucidate common cellular mechanisms. Such understanding is of fundamental scientific importance as neurodegenerative diseases represent one of the biggest health problems in our aging society, and uncovering molecular mechanisms of general validity is fundamental for the identification of new targets and development of rational therapies. The long-term goal of our group is to develop therapeutic and prophylactic anti-prion strategies. The overall objective we have is to study the cellular and molecular biology of prion infections and to use gained understanding for delineating novel targets for intervention. We have focused our attempts on two main strategies. One is the endogenous cellular clearance capacity for prions, the other one is to target the cellular isoform PrPc, which is a prerequisite for prion conversion and execution of neurodegeneration. It is our central hypothesis that it is feasible to interfere in prion propagation by increasing the cellular clearance for prions. Work in Aim 1 will substantiate our finding that prion clearance can be enhanced by compound-induced induction of autophagy, a basic cellular program for degradation and recycling. The proposed work intends to better understand the underlying molecular mechanisms and to validate the therapeutic and translational potential of this finding in vivo. Work in Aim 2 and 3 addresses cellular modifiers of prion formation. We have found that a basal level of autophagy is needed for establishing prion infection and we propose that autophagy represents the biological equivalent for the postulated disaggregase function in mammalian prion/prion-like biology. Our goal is to prove this at the cellular and molecular level. The rational for work in Aim 3 is that protein quality control mechanisms in the secretory pathway can directly influence prion conversion by determining on the quality of conversion substrates. We want to manipulate this by over-expressing folding and sorting proteins, in order to show that this represents a novel pathway counteracting prion propagation. Overall, our studies will provide mechanistic insights into basic cellular and molecular mechanisms which are relevant for neurodegenerative diseases and will result in novel targets for rational therapy against prion diseases and protein misfolding disorders.

With this award from the Major Research Instrumentation program (MRI) and the Chemistry Research Instrumentation program (CRIF), the University of Wyoming will acquire a time of flight mass spectrometer with matrix assisted laser desorption ionization technology and a liquid chromatograph (MALDI-TOF-LCMS). In general, mass spectrometry (MS) is one of the key analytical methods used to identify and characterize small quantities of chemical species embedded in complex matrices. A laser impinging on the inert matrix embedded with the sample, vaporizes and ionizes the sample. The ions pass into the mass spectrometer where the masses of the parent ion and its fragment ions are measured. This highly sensitive technique allows identification and determination of the structure of molecules in a complex mixture. An instrument with a liquid chromatograph provides additional identification power by separating mixtures of compounds before they are mass analyzed. The acquisition will strengthen the research infrastructure at the University and regional area. High school, undergraduate and graduate students from rural areas, including Native Americans, Hispanic/Latinos, women, and first-generation college students in Wyoming and surrounding Frontier states will benefit from training with this modern analytical technique.

The proposal is aimed at enhancing research and education at all levels, especially in areas such as (a) developing analytical methodologies for the rapid and precise analysis of biological and synthetic samples; (b) deciphering of protein folding dynamics and anti-freeze protein function; (c) developing photocatalytic organic materials; (d) developing glycosylated proteins; (e) elucidating cell messaging pathways; (f) studying fundamentals of evolutionary cell biology; and (g) developing clean energy sources.

? DESCRIPTION (provided by applicant): HIV infections have a significant impact on worldwide human health. Despite our inability to develop a highly effective vaccine to date, it is still widey believed that vaccination will be the best way to control the HIV pandemic. A highly effective vaccine might induce broadly neutralizing antibodies (bNAbs) that can neutralize diverse HIV isolates, analogous to those produced by some patients exhibiting long-term control over HIV infection. In addition to their ability to bind and/or neutralize different HIV variants, the Fc receptor (FcR)-mediated effector functions of these and other HIV-specific antibodies are essential for HIV control. Importantly, these effector functions can be influenced by the structures of the N-glycans found on all IgG Fc domains1. However, we need more comprehensive information on the impact of glycosylation profiles on bNAb functions in the overall immune response to HIV infection. Recent functional glycomic studies have generated interesting results, which not only support the idea that this research is necessary and important, but also demonstrate the need for further studies in this area. To date, functional glycomic studies have focused on a limited selection of bNAbs and bNAb glycoforms. Only one has involved in vivo analysis of different glycoforms15, none has assessed the impact of N-glycan structure on the ability of bNAbs to block mucosal transmission, and none has exploited a preclinical animal model permissive for HIV infection. Thus, we propose a comprehensive analysis of the impact of N-glycan structure on the in vitro and in vivo functions of representativ bNAbs in context of the overall immune response to HIV infection. Our functional glycomic approach will examine the broadest selection of bNAb glycoforms in the broadest set of assays used in any study reported to date. We will assess the impact of eight distinct N-glycosylation profiles on various antibody functions, including a wide variety of in vitro effector functions. We also will assess their overall abilities to protect against mucosal transmission of HIV in the hu-BLT mouse model. The results will reveal N-glycan-based mechanisms modulating the immune responses driven by two distinct HIV-specific bNAbs and identify one or more specific glycoforms as optimal targets for induction by an HIV vaccine and/or production for human therapeutic applications.

? DESCRIPTION (provided by applicant): HIV infections have a significant worldwide impact on human health. Despite our inability to develop a highly effective vaccine to date, it is still widey believed that vaccination could control the HIV pandemic. A highly effective vaccine might induce broadly neutralizing antibodies (bNAbs) that can neutralize diverse HIV isolates, analogous to those produced by some patients with long-term control over HIV infection1,2. This vaccine would probably also induce non-neutralizing antibodies (nNAbs) with potent effector functions that can reduce the risk of HIV infection, analogous to those produced by some subjects in the RV144 vaccine trial3-5. In parallel with vaccine development, it is thought that we can manufacture and passively administer bNAbs and/or nNAbs for post-exposure prophylaxis or as an adjunct to antiretroviral therapy, particularly against resistant viruses. The ability to broadly neutralize different HIV variants and induce potent effector functions are both critically important factors in the ability of HIV-specific antibodies (HIV-Abs) to control infection6. In tur, the potency of Fc receptor (FcR)-mediated effector functions is known to depend upon IgG N-glycan structure7. Unfortunately, we do not have comprehensive information on the impact of Fc glycosylation on relevant HIV-Ab functions in the overall immune response to HIV infection. Clearly, some recent studies have broken ground, provided important insights, and stimulated great interest in this area8-12. But, they also demonstrated the need for further analysis of a broader selection of glycoforms, of the impact of N-glycan structure on the ability of HIV- Abs to block mucosal transmission, and of the overall protective efficacy of different glycoforms in preclinical animal models permissive for HIV infection. We propose to achieve these goals in a more comprehensive analysis of the impact of N-glycan structure on the in vitro and in vivo functions of HIV-Abs in the overall immune response to HIV infection. We will use a VRC01 and A32 as models for functional glycomic studies designed to examine the broadest selection of glycoforms in the broadest set of assays used in any study reported to date. We will assess the impact of eight distinct N-glycosylation profiles on VRC01 and A32 functions, including viral neutralization (VRC01) and effector functions (both HIV-Abs). We also will assess the overall abilities of a selected subset of different VRC-01 and/or A32 glycoforms to protect against mucosal transmission of HIV in the hu-BLT mouse, a preclinical animal model system. The results will reveal N-glycan- based mechanisms modulating the immune responses driven by two functionally distinct HIV-Abs and identify one or more specific glycoforms as optimal targets for induction by an HIV vaccine and/or for recombinant manufacturing in advance of human therapeutic applications.

PROJECT SUMMARY Many biomedically significant proteins, including antibodies, cytokines, anticoagulants, blood clotting factors, and others are glycoproteins. Thus, there is a high demand for systems that can be used to produce recombinant glycoproteins for basic biomedical research and direct clinical applications. Unfortunately, few currently available recombinant protein production systems can produce higher eukaryotic glycoproteins with authentic, relatively homogeneous carbohydrate side chains at relatively low cost. The long-term objective of this proposal is to genetically engineer the silkworm, Bombyx mori, as a system that can fulfill these requirements for recombinant glycoprotein production. Numerous studies have shown that the silkworm silk gland, which has evolved for millions of years as a highly efficient silk protein production and secretion organ, can be engineered to efficiently produce and secrete recombinant proteins. However, transgenic silkworms have not yet been effectively used for recombinant glycoprotein production because the endogenous protein glycosylation pathways of the silk gland cannot properly glycosylate foreign, higher eukaryotic glycoproteins. The proposed research seeks to develop the silkworm as a novel system for recombinant human glycoprotein production by creating transgenic silkworms encoding a set of higher eukaryotic enzymes needed to ?humanize? the native silk gland protein N-glycosylation pathway and recombinant human N-glycoproteins of interest. Each transgene will be placed under the control of the tissue-specific Ser1 promoter to target its expression to the middle silk gland. To our knowledge, there is only one prior report of the effective genetic engineering of a protein glycosylation pathway in any multicellular animal, including B. mori. We will build upon our initial success of glycoengineering the protein N-glycosylation pathway of B. mori to significantly advance the use of the silk gland as a bioreactor for recombinant glycoprotein production and secretion. This will have a net positive effect on silk industries in both developed and underdeveloped countries worldwide, allowing value added products important for human health to be produced in an economically feasible manner in addition to the basic silk fiber, thereby significantly increasing the value of this important biomanufacturing platform. The Jarvis and Fraser laboratories have a demonstrated ability to perform this research as shown by their publication records. In addition, they have been productively collaborating on related projects for the past 15 years, generating a significant amount of relevant preliminary data. The complementary skills available in these two labs, their established working relationship, and the preliminary data obtained to date strongly suggest the proposed research can be successfully completed.