Stem Cells and Gene Delivery

Cell Therapies for Alzheimer's Disease

A promising genetically modified hMSC cell-based therapy is for the treatment of Alzheimer’s disease (AD).  AD afflicts more than 26 million people worldwide, is the leading cause of dementia in the elderly, and is one of the great health-care challenges of the 21st century.  AD mainly affects individuals 65 or older, and as the world population ages, the number of people affected by AD is expected to triple by 2050, with an estimated cost of $600 billion in the United States alone.    AD pathology is characterized by extracellular amyloid plaques, which consists of amyloid beta (Aβ) peptides, and intracellular neurofibrillary tangles (NFTs), which consists of hyperphosphorylated tau, in the brain. These hallmarks of AD, in turn, lead to inflammation and loss in neuronal function, which over time leads to atrophy in the neocortex resulting in severe and debilitating loss in cognitive functioning and death.    With no known cure for AD, scientists have developed many strategies targeted at all aspects of this complex, multifaceted disease with aims to halt the rising AD pandemic.  For instance, aggregation inhibitors and antibodies have been investigated as monotherapies in animal models and clinical trials for Aβ and NFT clearance, (Aduhelm, an Aβ targeted antibody, is the first FDA approved AD treatment in 18 years), while approaches that have focused on halting AD neurodegeneration and loss in cognitive functioning have mainly focused on delivering neurotrophic factors, such as human nerve growth factor (hNGF).  An alternative approach would be ex vivo genetic modification of hMSCs, as gene delivery could enhance hMSCs intrinsic therapeutic properties.

hMSCs have a host of unique properties that make hMSCs a viable cell type cell-based AD therapies, such as the ability to cross the blood-brain barrier (BBB), modulate inflammation, reduce Aβ plaques, and improve memory deficits in mouse models of AD.  Moreover, hMSCs have also been shown to produce molecular factors that would be beneficial for AD, such as neprilysin (NEP), an enzyme that has been shown to degrade Aβ.  However, in order to enhance hMSCs intrinsic AD therapeutic properties, such as increased NEP production, as well as induce expression of novel therapeutics targeted at the hallmarks of AD, such as a tau targeted phosphatase, protein phosphatase 2 regulatory subunit Balpha (PP2A/Bα), and a neurotrophic signal enhancer, glucagon-like peptide-1 (GLP-1), as each strategy alone has shown efficacy at halting AD pathological progression in a mouse model of AD-, the Pannier Lab is focused on developing an efficient hMSCs non-viral gene delivery system for therapeutic applications.

High-Throughput Approaches and Pharmacological Priming

Gene delivery is the delivery of exogenous genetic material to cells with the goal of altering molecular physiology to produce a phenotype change. Safe and efficient gene delivery has great potential to advance many applications in tissue engineering and regenerative medicine. Although viral vectors are efficient gene delivery vectors, clinical application of viral delivery are limited by safety issues. While nonviral delivery techniques are safer, they suffer from much lower efficiency.  Human mesenchymal stem cells (hMSCs) and other therapeutically relevant cells are especially difficult to nonvirally deliver nucleic acids to, and the barriers to successful ‘transfection’ are not well known. Therefore, the Pannier Lab seeks to investigate the molecular mechanisms that are important to nonviral gene delivery. A technique that can address certain barriers to the nonviral gene delivery process in hMSCs is simple addition of a pharmacological compound to the culture media, a process the Pannier Lab termed ‘priming’.  Specifically, we have demonstrated that an anti-inflammatory glucocorticoid drug known as dexamethasone can dramatically increase DNA transfection efficiency in hMSCs derived from multiple tissues and human donors, by modulating specific pathways related to cellular stresses induced by transfection.  Furthermore, we have expanded our hMSCs priming library by determining the ability of 707 FDA approved drugs from the National Institutes of Health Clinical Collection (NCC) to significantly modulate transfection compared to a vehicle control (VC) in adipose-derived hMSCs (AMSCs) from two donors. The priming drugs identified in the screen covered a diverse range of drug classes, such as glucocorticoids, flavonoids, stilbenoids, and antibiotics, that could significantly upregulate (glucocorticoids) or downregulate (flavonoids) hMSC transfection compared to a VC.

The priming compounds identified in our high-throughput screen of the NCC were primarily from drug classes that have yet to be used in hMSC transfection systems.  Therefore, the Pannier Lab is taking another high-throughput approach to identify possible molecular mechanisms of transfection modulation by the newly identified priming drug classes.  Drug Set Enrichment Analysis (DSEA), an online tool consisting of microarray data of endogenous gene expression changes following addition of select compounds, was employed to narrow down potential molecular targets and these targets expression level changes following priming and transfection were analyzed using RT-PCR and were further perturbed using small interfering RNA (siRNA).  The molecular mechanisms identified by the Pannier Lab from these newly identified priming compounds could help increase our understanding of the biology of transfection and guide others in the creation of more efficient nonviral gene delivery systems.

Mathematical Modeling

 Non-viral gene transfection involves a complex system of interacting cellular processes, each of which acts as a barrier towards successful and sustained expression of the delivered transgene. In an effort to understand the transfection-related process barriers in greater depth, our lab has developed and is working on the further improvement of a computational model based on telecommunications theory, wherein transfer of DNA to the nucleus is represented as a random packet-switched network. In this model, the DNA acts as a packet of information routed through various intermediate nodes, which represent the cellular barriers to successful transfection. As in a random packet-switched computer network, the routing of the DNA is seen as a stochastic and random process in which routing decisions are supported by packet characteristics and the network’s current state. Simulations involve the tracking of packets throughout individual intracellular networks within a population of cells over a 48-hour period. The model is currently capable of recapitulating transfection in HeLa cervical carcinoma cells. At this time, model parameters and architecture are being altered to represent the process in human mesenchymal stem cells, with the help of live-cell fluorescent imaging and algorithmic cell tracking.

Vector Optimization 

While our group has demonstrated that priming can significantly increase expression of a transgene in multiple donors of hMSCs, others, as well as the Pannier Lab, have demonstrated enhanced nonviral gene delivery to hMSCs by modifying the DNA vector.  For instance, minicircle DNA, which is a minimized eukaryotic expression cassette, has shown increased and sustained transgene expression in hMSCs when compared to plasmid DNA, as the minicircle vectors are devoid of bacterial elements, such as unmethylated cytosine-phosphate-guanine (CpG) sequences or antibiotic-resistance genes, that could activate an immune response and silence transgene expression.  An alternative approach to mitigating transgene silencing is reducing the length of the DNA sequence flanking the expression cassette.  For example, mini-intronic plasmid (MIP) DNA incorporates elements necessary for plasmid propagation and selection in bacteria in an engineered intron within a non-coding exon in the expression cassette, thereby reducing the length of DNA flanking the expression cassette, resulting in enhanced and sustained expression over plasmid DNA due to the reduced DNA vector size, as smaller sized DNA vectors have been shown to increase internalization and transgene expression compared to larger plasmids.   Moreover, different promoters incorporated into DNA vectors, such as cytomegalovirus (CMV) or elongation factor 1 alpha (EF1-α), have shown varying degrees of transgene expression enhancement in hMSCs at different time points after transfection, possibly due to expression of different transgenes (reporter or therapeutic) or to promoter silencing.  While various approaches have been taken to enhance nonviral gene delivery to hMSCs, these studies still do not achieve efficient transfection in hMSCs, possibly due to modulation of only one key aspect (e.g. DNA vector, promoter, cationic carrier, priming compound, and hMSC donor and tissue source).  Therefore, the focus of the work conducted in the Pannier Lab is to systematically compare these aspects of hMSC transfection systems as well as elucidate possible mechanisms of enhancements in order to guide future development of safe, easily implantable, and highly efficient nonviral gene delivery systems for hMSCs and hMSCs therapeutic applications.           

Physical Priming Strategies and Substrate-Mediated Delivery 

While chemical priming is the optimization of cell culture media to enhance transfection, physical priming is the optimization of cell culture surfaces for efficient transfection by tuning surface stiffness, topography, and chemistry.  In transfection with nucleic acid complexes, culture surface properties influence adsorption and release of complexes as well as cell adhesion, proliferation, and cytoskeletal dynamics that support complex endocytosis and intracellular transport.  We are developing two novel methods of modifying titanium surface chemistry and topography to support hMSC transfection, towards potential applications in both manufacturing of hMSC therapies as well as the design of gene activated dental and orthopedic implants.  First, in collaboration with the Leibniz Institute for Polymer Research in Dresden, Germany, we are testing a binary polymer brush system of poly(acrylic acid) and poly(N-isopropylacrylamide) as a platform for supporting the cell adhesion required for proliferation and transfection, while also potentially enabling temperature responsive cell detachment.  This modification of surface chemistry could improve the manufacturing of hMSC therapies by both priming transfection and enabling gentle cell release from the expansion surfaces that preserves cell membrane surface receptors and extracellular matrix.  Second, in collaboration with the Center for Electro-Optics and Functionalized Surfaces at the University of Nebraska-Lincoln, we are screening a wide range of micro and nano-scale surface topographies produced by femtosecond laser surface processing for supporting both cell adhesion as well as loading of complexes for surface-mediated delivery.  Screening surface-mediated transfection on these topographies can inform the development of gene activated bone implants that have decreased failure rates and increased implant lifetimes.

Using CRISPR/Cas9 to Control Gene Expression and Stem Cell Differentiation

Since being first described in 2012 as a powerful and flexible genome editing tool able to target virtually any DNA sequence with a complementary guide RNA (gRNA), CRISPR technologies have become the most widely adopted molecular biology tool. In addition to editing genomic sequences, CRISPR systems have also been engineered to provide a diverse toolbox for targeted engineering of many facets of biological systems. Specifically, CRISPR systems can be used to activate and/or repress transcription of target genes for precise control over gene expression. One of those systems makes use of dCas9 fused to a histone acetyltransferase (i.e. p300) to edit the epigenetic code (i.e. histone acetylation) and subsequently activate target gene expression. However, clinical application of these CRISPR technologies are limited by delivery challenges. Viral delivery of CRISPR systems is efficient, but suffers from safety issues, while safer nonviral delivery is too inefficient for most clinically-relevant applications. Therefore, the Pannier Lab is working to develop more efficient nonviral delivery strategies for use in clinically-relevant cells like human mesenchymal stem cells (hMSCs). Early work has shown that our optimized protocol can deliver the dCas9-p300 system to hMSCs and successfully activate expression of target genes. Since control of gene expression of hMSCs and other clinically relevant cell types could enable more precise differentiation of cells to unique phenotypes, we aim to further develop nonviral CRISPR-based strategies for novel tissue engineering and regenerative medicine applications.