BIRMINGHAM, AL, United States

Agenta Biotechnologies, Inc.

www.agentabiotechnologies.com
BIRMINGHAM, AL, United States

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Ellis A.L.,Agenta Biotechnologies, Inc. | Pan W.,Agenta Biotechnologies, Inc. | Pan W.,Southern Research Institute | Yang G.,Agenta Biotechnologies, Inc. | And 6 more authors.
BMC Biotechnology | Year: 2010

Background: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology.Results: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high MrrhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kDof 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.Conclusions: With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles. © 2010 Ellis et al; licensee BioMed Central Ltd.


DeCarlo A.A.,Agenta Biotechnologies, Inc. | Belousova M.,Agenta Biotechnologies, Inc. | Ellis A.L.,Agenta Biotechnologies, Inc. | Petersen D.,Agenta Biotechnologies, Inc. | And 5 more authors.
BMC Biotechnology | Year: 2012

Background: Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo.Results: Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™).Conclusions: A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective. © 2012 DeCarlo et al.; licensee BioMed Central Ltd.


Dooley T.P.,Agenta Biotechnologies, Inc. | Ellis A.L.,Agenta Biotechnologies, Inc. | Belousova M.,Agenta Biotechnologies, Inc. | Petersen D.,Agenta Biotechnologies, Inc. | DeCarlo A.A.,Agenta Biotechnologies, Inc.
Journal of Biomaterials Science, Polymer Edition | Year: 2013

High density chitosan membranes were produced via a novel manufacturing process and used as implantable resorbable surgical membranes. The innovative method utilizes the following three sequential steps: (1) casting an acidic chitosan solution within a silicon mold, followed by freezing; (2) neutralizing the frozen acidic chitosan solution in alkaline solution to facilitate polymerization; and (3) applying coincident compression-dehydration under a vacuum. Resulting membranes of 0.2-0.5 mm thickness have densities as high as 1.6g/cm3. Inclusion of glycerol prior to the compression-dehydration step provides additional physical and clinical handling benefits. The biomaterials exhibit tensile strength with a maximum load as high as 10.9 N at 2.5mm width and clinically relevant resistance to suture pull-out with a maximum load as high as 2.2 N. These physical properties were superior to those of a commercial reconstituted collagen membrane. The dense chitosan membranes have excellent clinical handling characteristics, such as pliability and 'memory' when wet. They are semipermeable to small molecules, biodegradable in vitro in lysozyme solution, and the rates of degradation are inversely correlated to the degree of deacetylation. Furthermore, the dense chitosan membranes are biocompatible and resorbable in vivo as demonstrated in a rat oral wound healing model. The unique combination of physical, in vitro, in vivo, and clinical handling properties demonstrate the high utility of dense chitosan membranes produced by this new method. The materials may be useful as surgical barrier membranes, scaffolds for tissue engineering, wound dressings, and as delivery devices for active ingredients. © 2012 Taylor & Francis.


Patent
Agenta Biotechnologies, Inc. | Date: 2011-12-22

A composition of exceptionally dense chitosan and a novel method for producing the dense chitosan structure have been described. The novel production method employs coincident compression and vacuum on a neutralized chitosan polymer that results in an exceptionally dense chitosan film or membrane material. The dense chitosan film or membrane composition possesses multiple physical and clinically appealing qualities for a variety of medical applications on or in animals, mammals, or humans.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 156.79K | Year: 2010

DESCRIPTION (provided by applicant): In chronic wounds, numerous factors contribute to the diminished healing. Wound healing constituents, including heparan sulfate proteoglycans and numerous growth factors, are essential to normal skin wound healing. Several key growth factors such as FGF, VEGF, and PDGF require heparan sulfate-like polymers as co- receptors or co-activators for growth factor activity. Agenta Biotechnologies, Inc. has developed expertise and IP to generate heparan sulfate in situ, enabling secretion of a customized heparan sulfate polymer with a sulfation pattern specific for the wound environment. It is the objective of this project to develop a skin wound co-therapeutic that provides both a growth factor together with the critical co-stimulatory heparan sulfate proteoglycan to the healing wound for accelerated wound closure. Together with vascular endothelial growth factor, isoform 165 (VEGF165) we will deliver this co-therapeutic to full thickness excisional wounds on the dorsum of normal (Aim 1) and diabetic rats (Aim 2) via a chitosan dressing. Plasmid DNA expression constructs of the proteoglycan and the VEGF165 transgenes are characterized and ready for this proposed project. Alternatively, responses to replication-deficient adenovirus delivery of the co-therapeutic will be measured. The primary outcome measurement will be wound closure rates. Secondary outcome measurements of wound integrity include vascularization, collagen content, reepitheliazation, and granulation. Planned phase 2 work includes investigation of co-delivering FGF and PDGF with proteoglycan transgenes, as well as developing recombinant-based proteoglycan/growth factor therapeutics. Wound healing and tissue regeneration have begun a new era where the augmentation of bandages and dressings with key biological cell activators is showing success and gaining approval. Agenta Biotechnologies, Inc. has established themselves as leaders in manipulating proteoglycans for therapeutic use and is well-positioned to advance this novel therapeutic approach addressing the serious problem of impaired wound healing. PUBLIC HEALTH RELEVANCE: Upregulation of growth factors and heparan sulfate proteoglycans, especially those shown to be involved in wound healing, in the wound bed could enhance wound closure rates and wound integrity, especially needed for patients with impaired wound healing, such as diabetics. The first domain of perlecan, decorated with 3 heparan sulfate chains, and vascular endothelial growth factor, shown to be effective in wound healing are rational choices for this proposed co-therapeutic. This project will measure delivery dose, wound closure rates, and wound integrity in both normal and diabetic rat models.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 157.07K | Year: 2011

DESCRIPTION (provided by applicant): Advancement in wound management or tissue repair has been limited by the complexities, costs and limited efficacy of purified recombinant growth factors. Further, the combination of these recombinant proteins for moresophisticated approaches is complicated by the need for separate biologics. Optimally, we can move to a single biologic DNA pro-drug that provides for the generation of multiple growth factors that will work in concert with the required heparan sulfate co-receptor. Such a DNA pro-drug could be applied locally within a graft, bandage or gel to different tissue types including bone and skin, and the DNA pro- drug will have relatively greater stability than a comparable protein class of drugs making it ideal for field use. The platform DNA pro-drug could be modified easily will have relatively greater stability than a comparable protein class of drugs making it ideal for field use with greater affordability and wide-spread access. We will begin by testing pro-drugs expressing 2 or 3 growth factors designed for bone reconstruction and repair using a calcium phosphate graft scafold for sustained release. A significant aspect of this proposed technology is the co-expression of the required growth factor co-receptorfor growth factor activity - a heparan sulfate proteoglycan. We have identified an elegant mechanism to provide this required growth factor co-receptor using a transgene that generates a site-specific heparan sulfate proteoglycan. DNA pro- drug delivery of this heparan sulfate transgene significantly enhanced growth factor activity and bone healing in the maxilla, providing a 3-fold dose enhancement of BMP-2 activity in preliminary experiments. Four US patents describing the essence of this technology havebeen awarded to Agenta Biotechnologies, Inc. The Specific Aims of this project are as follows: 1) create, characterize, and pre-clinically test efficacy of an expression plasmid that expresses both the heparan sulfate co-receptor and BMP-2; and 2) create,characterize, and pre-clinically test efficacy of an expression plasmid that expresses VEGF165 along with the heparan sulfate co-receptor and BMP-2, testing the additional need for rapid vascularization of a large avascular synthetic bone graft in a large, critical size graft area. In vivo, a well-characterized maxillary ridge augmentation model will deliver the experimental DNA pro-drugs on an approved calcium phosphate graft material to the surface of the bone with the goal of building new and unprecedented levels of additional bone that is fully integrated with the existing jaw. Primary in vitro outcomes include assessment of expression levels, and biologic function. Functional assays will measure osteoblast mineralization rates, cell proliferation, andendothelial cell vasculogenesis. Primary in vivo outcomes will be the volume and relative density of new bone or osteoid as measured using histomorphometric analysis. Secondary outcomes include histomorphometric assessments of inflammation, molecular evidence for in situ expression of the transgenes, and a pathology analysis. PUBLIC HEALTH RELEVANCE: Advancement of biologic therapeutics and the potential benefits they can provide in wound management and tissue regeneration has been limited by the complexities of manufacturing, regulation, stability, and partial efficacy of purified recombinant growth factors. The combination of these recombinant proteins for more sophisticated approaches to wound healing is complicated by the need for separate biologics. Optimally, we can move to a single biologic DNA pro-drug that provides for the generation of multiple growth factors together with the required heparan sulfate co-receptor for growth factors. In essence, this technology proposes multiple drug activities working in concert for tissue healing, regeneration, or remodeling, to be generated from a single pro-drug. Such a DNA pro-drug could be applied locally within a graft, bandage or gel for healing, regeneration, or remodeling of different tissue types including bone and skin, with engineered tissue-specific on-off controls for different growth factors. The platform DNA pro- drug could be modified easily and the DNA pro-drug will have relatively greater stability than a comparable protein class of drugs making it ideal for field use with greater affordability and wide-spread access. We will begin in this phase 1 project by testing pro-drugs expressing 2 or 3 biologics designed for bone reconstruction and repair. A significant aspect of this proposed technology is the co-expression of the required growth factor co-receptor for growth factor activity - a heparan sulfate proteoglycan. The proposed technology is a rational and timely approach to establish a new generation of therapeutics capable of cost-effectively and practically providing multiple biological drug activities for advanced wound management or tissue repair.


PubMed | Agenta Biotechnologies, Inc.
Type: | Journal: BMC biotechnology | Year: 2012

Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo.Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse).A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.


Trademark
Agenta Biotechnologies, Inc. | Date: 2011-07-14

Baseball caps and hats; T-shirts.


Agenta Biotechnologies, Inc. | Entity website

Commercialization plan Value, Expected Outcomes, and Impact. Chronic ulcers can be debilitating, painful, become infected to varying degrees, often require surgical therapy, and, if peripherally located on the leg or foot, can lead to amputation ...


Agenta Biotechnologies, Inc. | Entity website

PLATFORM TECHNOLOGY & PRODUCTS: Our platform technology is based upon high density chitosan membranes that are typically < 0.8 mm in thickness, and are intended for use in dental and medical surgical procedures ...

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