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Biomedical subjects

Daniel G Anderson

Publications and source records attributed to Daniel G Anderson.

17 recordsLinked to original sources

The Use of Deep Learning in RNA Therapeutic Development.

Ribonucleic acid (RNA)-based therapeutics have emerged as promising methods of disease treatment due to their ability to target the human genome and influence protein production, their versatility, and their relative lack of toxicity compared to other gene therapies. However, the RNA therapeutic design space is extremely large, encompassing multiple variables, including codon identities, secondary structure, and design of specific regions. RNA therapeutic optimization is difficult due to the impracticality of exploring such a vast design space experimentally. To address this limitation, deep learning methods have been employed to optimize RNA therapeutic development. In this review, we examine the application of deep learning models across three key aspects of RNA therapeutic development (RNA structure prediction, CRISPR activity, and RNA delivery), highlighting major contributions in these fields and analyzing how deep learning model architectures could affect model performance. We then discuss challenges associated with using deep learning for RNA therapeutics, such as computational and data limitations. Finally, we offer perspectives on areas for future exploration, such as emerging model architectures and methods of integration with more advanced high-throughput screening techniques. Ultimately, this review provides an overview of how deep learning is used in RNA therapeutic development and how it can evolve in the future.

Deep Learning↗

Synthesis of poly(beta-amino ester)s with thiol-reactive side chains for DNA delivery.

The safe and efficient delivery of DNA remains the major barrier to the clinical application of non-viral gene therapy. Here, we present novel, biodegradable polymers for gene delivery that are capable of simple graft modification and demonstrate the ability to respond to intracellular conditions. We synthesized poly(beta-amino ester)s using a new amine monomer, 2-(pyridyldithio)-ethylamine (PDA). These cationic, degradable polymers contain pyridyldithio functionalities in the side chains that react with high specificity toward thiol ligands. This reactivity is demonstrated using both mercaptoethylamine (MEA) and the thiol peptide RGDC, a ligand that binds with high affinity to certain integrin receptors. These two polymer derivatives displayed strong DNA binding as determined using electrophoresis and dye exclusion assays. In addition, the MEA-based polymer and plasmid DNA were shown to self-assemble into cationic complexes with effective diameters as low as 100 nm. Furthermore, this DNA binding ability was substantially reduced in response to intracellular glutathione concentrations, which may aid in DNA unpackaging inside the cell. These complexes also displayed low cellular toxicity and were able to mediate transfection at levels comparable to PEI in human hepatocellular carcinoma cells. These results suggest that PDA-based poly(beta-amino ester)s may serve as a modular platform for polymer-mediated gene delivery.

Biocompatible Materials↗

Biomaterial microarrays: rapid, microscale screening of polymer-cell interaction.

The identification of biomaterials that induce optimal gene expression patterns and allow for appropriate levels of cellular attachment is of central importance in tissue engineering and cell therapy. Herein, we describe the creation of cell-compatible, biomaterial microarrays, that allow rapid, microscale testing of biomaterial interactions with cells. As proof of principle, we simultaneously characterized over 3456 human mesenchymal stem cell (hMSC)-biomaterial composite interactions, and describe preliminary studies on the utility of these arrays with a neural stem cell line (NSC), and primary articular chondrocytes.

Biocompatible Materials↗

Direct patterning of mammalian cells onto porous tissue engineering substrates using agarose stamps.

This paper describes simple, inexpensive, and potentially generic methodology for generating patterns of mammalian cells on porous scaffolds for tissue engineering using replica printing. Circular patterns (diameter: 200, 700, and 1000 microm) of human osteoblasts were transferred directly from topographically patterned agarose stamps onto porous hydroxyapatite scaffolds or onto fibronectin-coated glass slides. The use of hydrogel stamps provided a "wet", biocompatible surface and maintained the viability of cells adsorbed on stamps during the patterning process. Stamps inked once with suspensions of cells allowed the repeated patterning of substrates. Direct stamping of human osteoblasts (and, potentially other mammalian cells) can be used to control the size, spacing, and geometry of patterns of cells printed on porous tissue engineering substrates. This approach may find use in controlling the spatial invasion of scaffolds, promoting the hierarchical organization of cells, and in controlling cell-cell interactions as a step in preservation of phenotypes of cells.

Actins↗

Structure/property studies of polymeric gene delivery using a library of poly(beta-amino esters).

Here we describe the synthesis and characterization of a library of 486 second-generation poly(beta-amino esters). To understand better the structure/property relationships governing polymeric gene delivery, we synthesized polymers with 70 different primary structures, at 6 to 12 different molecular weights, using monomers previously identified as common to effective gene delivery polymers. This library was characterized by (1) molecular weight, (2) particle size upon complexation with DNA, (3) surface charge upon complexation with DNA, (4) optimal polymer/DNA ratio, and (5) transfection efficiency. In this library, polymers with 20 of the 70 primary structures possess transfection efficiencies as good as or better than one of the best commercially available lipid reagents, Lipofectamine 2000. In general, the most effective polymers condense DNA into sub-150-nm complexes with positive surface charge. Among this group, the 2 most effective polymers condensed DNA to the smallest particle sizes (71 and 79 nm). Interestingly, the top 9 polymers were all formed from amino alcohols, and the structure of the 3 top performing polymers differs by only one carbon. This convergence in structure of the top performing polymers suggests a common mode of action and provides a framework with which future polymers can be designed.

Animals↗

Beta-amino ester polymers facilitate in vivo DNA transfection and adjuvant plasmid DNA immunization.

Increased in vivo expression of intramuscularly delivered plasmid DNA will be essential for clinical success in gene therapy and plasmid DNA vaccination. We screened polymers from a library of beta-amino esters for their ability to augment transgene expression as measured by beta-galactosidase activity and cellular immune responses. Among the candidates identified in this screen, poly[(1,6-di(acryloxyethoxy)hexane)-co-(4-aminobutanol)] enhanced plasmid DNA transgene expression by sevenfold (P=0.0001) and its immunogenicity by 70% (P=0.03). We found that polymers with moderately hydrophobic backbones and terminal alcohol groups facilitated transfection most effectively in vivo. We also observed a log-linear correlation (R2=0.93) between peak cellular immune responses and transgene activity in all evaluated polymer-plasmid DNA formulations, clarifying the relationship between immunogenicity and the quantity of expressed antigen.

Animals↗

A polymer library approach to suicide gene therapy for cancer.

Optimal gene therapy for cancer must (i) deliver DNA to tumor cells with high efficiency, (ii) induce minimal toxicity, and (iii) avoid gene expression in healthy tissues. To this end, we generated a library of >500 degradable, poly(beta-amino esters) for potential use as nonviral DNA vectors. Using high-throughput methods, we screened this library in vitro for transfection efficiency and cytotoxicity. We tested the best performing polymer, C32, in mice for toxicity and DNA delivery after intratumor and i.m. injection. C32 delivered DNA intratumorally approximately 4-fold better than one of the best commercially available reagents, jetPEI (polyethyleneimine), and 26-fold better than naked DNA. Conversely, the highest transfection levels after i.m. administration were achieved with naked DNA, followed by polyethyleneimine; transfection was rarely observed with C32. Additionally, polyethyleneimine induced significant local toxicity after i.m. injection, whereas C32 demonstrated no toxicity. Finally, we used C32 to deliver a DNA construct encoding the A chain of diphtheria toxin (DT-A) to xenografts derived from LNCaP human prostate cancer cells. This construct regulates toxin expression both at the transcriptional level by the use of a chimeric-modified enhancer/promoter sequence of the human prostate-specific antigen gene and by DNA recombination mediated by Flp recombinase. C32 delivery of the A chain of diphtheria toxin DNA to LNCaP xenografts suppressed tumor growth and even caused 40% of tumors to regress in size. Because C32 transfects tumors locally at high levels, transfects healthy muscle poorly, and displays no toxicity, it may provide a vehicle for the local treatment of cancer.

Animals↗

pH-triggered microparticles for peptide vaccination.

Improving vaccine delivery to human APCs is a way to increase the CTL response to vaccines. We report the use of a novel pH-triggered microparticle that exploits the ability of APCs to cross-present MHC I-restricted Ags that have been engulfed in the low pH environment of the phagosome. A model MHC class I-restricted peptide Ag from the influenza A matrix protein was encapsulated in spray-dried microparticles composed of dipalmitoylphosphatidylcholine and the pH-sensitive polymethacrylate Eudragit E100. Release of the peptide from the particle was triggered by a drop in pH to the acidity normally found in the phagosome. The particles were efficiently phagocytosed by human monocytes and dendritic cells with minimal cellular toxicity and no functional impairment. Encapsulation of the peptide in the microparticles resulted in efficient presentation of the peptide to CD8(+) T cells by human dendritic cells in vitro, and was superior to unencapsulated peptide or peptide encapsulated in an analogous pH-insensitive particle. Vaccination of human HLA-A*0201 transgenic mice with peptide encapsulated in pH-triggering microparticles resulted in priming of CTL responses. These microparticles can be modified to coencapsulate a range of adjuvants along with the Ag of interest. Encapsulation of MHC I epitopes in pH-triggered microparticles increases Ag presentation and may improve CD8(+) T cell priming to peptide vaccines against viruses and cancer.

1,2-Dipalmitoylphosphatidylcholine↗

Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines.

Current nonviral genetic vaccine systems are less effective than viral vaccines, particularly in cancer systems where epitopes can be weakly immunogenic and antigen-presenting cell processing and presentation to T cells is down-regulated. A promising nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect plasmid payloads and target them to phagocytic antigen-presenting cells. However, conventional microparticle formulations composed of poly lactic-co-glycolic acid take too long to release encapsulated payload and fail to induce high levels of target gene expression. Here, we describe a microparticle-based DNA delivery system composed of a degradable, pH-sensitive poly-beta amino ester and poly lactic-co-glycolic acid. These formulations generate an increase of 3-5 orders of magnitude in transfection efficiency and are potent activators of dendritic cells in vitro. When used as vaccines in vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells.

Animals↗

Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells.

Identification of biomaterials that support appropriate cellular attachment, proliferation and gene expression patterns is critical for tissue engineering and cell therapy. Here we describe an approach for rapid, nanoliter-scale synthesis of biomaterials and characterization of their interactions with cells. We simultaneously characterize over 1,700 human embryonic stem cell-material interactions and identify a host of unexpected materials effects that offer new levels of control over human embryonic stem cell behavior.

Biocompatible Materials↗

Parallel synthesis and biophysical characterization of a degradable polymer library for gene delivery.

We recently reported the parallel synthesis of 140 degradable poly(beta-amino esters) via the conjugate addition of 20 primary or secondary amine monomers to seven different diacrylate monomers. To explore possible structure/function relationships and further characterize this class of materials, we investigated the ability of each DNA-complexing polymer to overcome important cellular barriers to gene transfer. The majority of vectors were found to be uptake-limited, but complexes formed from polymers B14 and G5 displayed high levels of internalization relative to "naked" DNA (18x and 32x, respectively). Effective diameter and zeta potential measurements indicated that, in general, small particle size and positive surface charge led to higher internalization rates. Of the 10 DNA/polymer complexes with the highest uptake levels, all had effective diameters less than 250 nm and nine had positive zeta potentials. Lysosomal trafficking was investigated by measuring the pH environment of delivered DNA. Complexes prepared with polymers G5, G10, A13, B13, A14, and B14 were found to have near neutral pH measurements, suggesting that they were able to successfully avoid trafficking to acidic lysosomes. This work highlights the value of parallel synthesis and screening approaches for the discovery of new polymers for gene delivery and the elucidation of structure/function relationships for this important class of materials.

3T3 Cells↗

pH-triggered release of macromolecules from spray-dried polymethacrylate microparticles.

PURPOSE: pH-triggered microparticles release their therapeutic payloads at acidic pH (e.g., in the phagosome), making intracellular drug delivery more efficient. Here we modify lipid-based microparticles that are safe and efficacious in nerve and brain and are potentially inhalable, making them pH-triggerable by incorporating an acid-soluble polymethacrylate, Eudragit E100 (E100). METHODS: Microparticles were produced by spray-drying and characterized by electron microscopy, Coulter counting, density measurement, and release kinetics of fluorescently labeled proteins. In addition, biocompatibility and cellular uptake were observed in rats. RESULTS: Microparticles were spheroids 3 to 5 microm in diameter with densities of 0.12 to 0.25 g/L. Microparticles with 20% (w/w) or more E100 demonstrated slow release of fluorescently labeled proteins at pH 7.4 but rapid release at pH 5. pH-triggerability was maintained for over 2 weeks in solution. Protein loadings of 0.2-20% (w/w) were pH-triggerable. Histologic examination of particles in rat connective tissue near nerve and muscle demonstrated biocompatibility aside from muscle edema in the cell layers adjacent to the particles and a localized inflammatory reaction with macrophages laden with microparticles. CONCLUSIONS: Microparticles containing E100 were pH-triggerable for many days and were taken up by macrophages, suggesting that they may be useful for intracellular drug delivery.

Acrylates↗

Synthesis of poly(beta-amino ester)s optimized for highly effective gene delivery.

Several families of synthetic polymers, including degradable poly(beta-amino ester)s, have been previously shown to effectively mediate gene transfer. However, the combined impact of potentially significant factors-such as polymer molecular weight, polymer chain end-group, and polymer/DNA ratio-on different gene transfer properties has yet to be systematically investigated. The elucidation of these relationships may aid in the design of nonviral vectors with greatly enhanced transfection properties. To examine these factors, two distinct poly(beta-amino ester) structures, Poly-1 and Poly-2, were generated by adding 1,4-butanediol diacrylate and 1,6-hexanediol diacrylate, respectively, to 1-aminobutanol. Twelve unique versions of each structure were synthesized by varying amine/diacrylate stoichiometric ratios, resulting in polymers with either amine or acrylate end-groups and with molecular weights ranging from 3350 to 18000. Using high throughput methods, all polymers were tested in quadruplicate at nine different polymer/DNA ratios ranging from 10:1 w/w to 150:1 w/w. Through the optimization of molecular weight, polymer chain end-group, and polymer/DNA ratio, these polymers successfully mediated gene transfer at levels that surpassed both PEI and Lipofectamine 2000 in vitro.

Animals↗

Biodegradable polymeric vectors for gene delivery to human endothelial cells.

Endothelial cells are an important cell type to both cardiovascular disease and cancer, as they play critical roles in vascular function and angiogenesis. However, effective and safe gene delivery to primary endothelial cells in the presence of serum proteins is known to be particularly challenging. A library of biodegradable poly(beta-amino esters) was synthesized for use as potential vectors. Promising vectors were optimized for high efficacy and low cytotoxicity to human umbilical vein endothelial cells (HUVECs) in serum. Vector parameters including polymer type, polymer weight, and DNA loading were varied, and biophysical properties including particle size, zeta potential, and particle stability over time were studied. While many of the poly(beta-amino ester) vectors have similar biophysical properties in the presence of buffer, their biophysical properties changed differentially in the presence of serum proteins, and the properties of these serum-interacting particles correlated to transfection efficacy. Leading poly(beta-amino ester) vectors were found to transfect HUVECs in the presence of serum significantly higher (47 +/- 9% positive, n = 10) than the best commercially available transfection reagents including jetPEI (p < 0.001) and Lipofectamine 2000 (p < 0.01). These results demonstrate the potential of a new class of biomaterials, poly(beta-amino esters), for effective human endothelial cell gene therapy.

Absorbable Implants↗

Degradable poly(amino alcohol esters) as potential DNA vectors with low cytotoxicity.

The synthesis of a new degradable polymer system, poly(amino alcohol esters) and the resulting polymers' potential for use in gene transfection vectors are reported. The polymerization proceeded in a one step reaction from commercially available bis(secondary amines) monomers (N,N'-dimethyl-1,3-propanediamine and N,N'-dimethyl-1,6-hexanediamine, respectively) through nucleophilic addition to the diglycidyl ester of dicarboxylic acid (diglycidyl adipate). Poly(amino alcohol ester) 1 and 2 were synthesized with a yield of 89% and 91% with Mn = 24,800 and Mn = 36,400, respectively. Poly(amino alcohol ester) 1 degraded hydrolytically in phosphate buffer at pH 7.4 with a half-life of approximately 5 days. Both polymers readily self-assembled with plasmid DNA into nanometer-sized DNA/polymer complexes less than 180 nm diameter and are significantly less cytotoxic than the commonly used DNA delivery polymer, poly(ethylene imine) (PEI).

3T3 Cells↗