14 research outputs found
hPSC-Derived Striatal Cells Generated Using a Scalable 3D Hydrogel Promote Recovery in a Huntington Disease Mouse Model.
Increasing Cancer-Specific Gene Expression by Targeting Overexpressed α<sub>5</sub>β<sub>1</sub> Integrin and Upregulated Transcriptional Activity of NF-κB
We developed a modular multifunctional
nonviral gene delivery system
by targeting the overexpressed cancer surface receptor α<sub>5</sub>β<sub>1</sub> integrin and the upregulated transcriptional
activity of the cancer resistance mediating transcription factor NF-κB,
thereby introducing a new form of transcriptional targeting. NF-κB
regulated therapy can improve specificity of gene expression in cancer
tissue and also may offset NF-κB mediated cancer resistance.
We delivered a luciferase gene under the control of an NF-κB
responsive element (pNF-κB-Luc) encapsulated in a PR_b peptide
functionalized stealth liposome that specifically targets the α<sub>5</sub>β<sub>1</sub> integrin and achieved increased gene expression
in DLD-1 colorectal cancer cells compared to BJ-fibroblast healthy
cells <i>in vitro</i>. The multitargeted system was also
able to differentiate between cancer cells and healthy cells better
than either of the individually targeted systems. In addition, we
constructed a novel cancer therapeutic plasmid by cloning a highly
potent diphtheria toxin fragment A (DTA) expressing gene under the
control of an NF-κB responsive element (pNF-κB-DTA). A
dose-dependent reduction of cellular protein expression and increased
cytotoxicity in cancer cells was seen when transfected with PR_b functionalized
stealth liposomes encapsulating the condensed pNF-κB-DTA plasmid.
Our therapeutic delivery system specifically eradicated close to 70%
of a variety of cancer cells while minimally affecting healthy cells <i>in vitro</i>. Furthermore, the modular nature of the nonviral
design allows targeting novel pairs of extracellular receptors and
upregulated transcription factors for applications beyond cancer gene
therapy
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cAMP and EPAC Signaling Functionally Replace OCT4 During Induced Pluripotent Stem Cell Reprogramming
The advent of induced pluripotent stem cells--generated via the ectopic overexpression of reprogramming factors such as OCT4, SOX2, KLF4, and C-MYC (OSKM) in a differentiated cell type--has enabled groundbreaking research efforts in regenerative medicine, disease modeling, and drug discovery. Although initial studies have focused on the roles of nuclear factors, increasing evidence highlights the importance of signal transduction during reprogramming. By utilizing a quantitative, medium-throughput screen to initially identify signaling pathways that could potentially replace individual transcription factors during reprogramming, we initially found that several pathways--such as Notch, Smoothened, and cyclic AMP (cAMP) signaling--were capable of generating alkaline phosphatase positive colonies in the absence of OCT4, the most stringently required Yamanaka factor. After further investigation, we discovered that cAMP signal activation could functionally replace OCT4 to induce pluripotency, and results indicate that the downstream exchange protein directly activated by cAMP (EPAC) signaling pathway rather than protein kinase A (PKA) signaling is necessary and sufficient for this function. cAMP signaling may reduce barriers to reprogramming by contributing to downstream epithelial gene expression, decreasing mesenchymal gene expression, and increasing proliferation. Ultimately, these results elucidate mechanisms that could lead to new reprogramming methodologies and advance our understanding of stem cell biology
Transfection Mechanisms of Polyplexes, Lipoplexes, and Stealth Liposomes in α<sub>5</sub>β<sub>1</sub> Integrin Bearing DLD‑1 Colorectal Cancer Cells
Receptor
targeted, PEGylated transfection agents can improve stability
and delivery specificity of current cationic lipid and polymer based
nonviral gene delivery vehicles, but their mode of transfection is
poorly understood. We therefore investigated the transfection mechanisms
of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE) lipoplexes, branched
polyethylenimine (bPEI) polyplexes, and bPEI encapsulated in either
PEGylated (stealth) nontargeted liposomes or PR_b peptide (targeted
to α<sub>5</sub>β<sub>1</sub> integrin) functionalized
stealth liposomes in DLD-1 colorectal cancer cells in vitro with gene
expression assays, flow cytometry and confocal microscopy. DOTAP/DOPE
and PR_b functionalized stealth liposomes mediated higher gene expression
compared to nontargeted stealth liposomes and bPEI. However DOTAP/DOPE
was internalized slowly leading to lower levels of DNA uptake. In
contrast, despite high internalization of bPEI polyplexes, gene expression
levels were low as DNA was unable to escape from the endosomes. Nontargeted
stealth liposomes also mediated low gene expression due to low amounts
of DNA internalized and slow internalization kinetics. PR_b functionalized
stealth liposomes struck an optimal balance among these transfection
agents with efficient transfection arising from fast integrin mediated
internalization kinetics, high amounts of DNA uptake, and endosomal
escape. We found α<sub>5</sub>β<sub>1</sub> integrin to
be a valuable target for gene delivery and that the caveolar endocytic
pathway may offer an advantage to receptor targeted PEGylated transfection
agents in DLD-1 cells
Preparation and Characterization of Liposome-Encapsulated Plasmid DNA for Gene Delivery
The success of common nonviral gene
delivery vehicles, lipoplexes
and polyplexes, is limited by the toxicity and instability of these
charged molecules. Stealth liposomes could provide a stable, safe
alternative to cationic DNA complexes for effective gene delivery.
DNA encapsulations in three stealth liposomal formulations prepared
by thin film, reverse phase evaporation, and asymmetric liposome formation
were compared, and the thin film method was found to produce the highest
yields of encapsulated DNA. A DNA quantification method appropriate
for DNA encapsulated within liposomes was also developed and verified
for accuracy. The effect of initial lipid and DNA concentrations on
the encapsulation yield and fraction of DNA-filled liposomes was evaluated.
Higher encapsulation yields were achieved by higher lipid contents,
while a higher fraction of DNA-filled liposomes was produced by either
lower lipid content or higher DNA concentration. Control of these
parameters allows for the design of gene delivery nanoparticles with
high DNA encapsulation yields or higher fraction of DNA-filled liposomes
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CRISPR-Cas9-Mediated Genome Editing Increases Lifespan and Improves Motor Deficits in a Huntington's Disease Mouse Model.
Huntington's disease (HD) is a currently incurable and, ultimately, fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion within exon 1 of the huntingtin (HTT) gene, which results in the production of a mutant protein that forms inclusions and selectively destroys neurons in the striatum and other adjacent structures. The RNA-guided Cas9 endonuclease from CRISPR-Cas9 systems is a versatile technology for inducing DNA double-strand breaks that can stimulate the introduction of frameshift-inducing mutations and permanently disable mutant gene function. Here, we show that the Cas9 nuclease from Staphylococcus aureus, a small Cas9 ortholog that can be packaged alongside a single guide RNA into a single adeno-associated virus (AAV) vector, can be used to disrupt the expression of the mutant HTT gene in the R6/2 mouse model of HD following its in vivo delivery to the striatum. Specifically, we found that CRISPR-Cas9-mediated disruption of the mutant HTT gene resulted in a ∼50% decrease in neuronal inclusions and significantly improved lifespan and certain motor deficits. These results thus illustrate the potential for CRISPR-Cas9 technology to treat HD and other autosomal dominant neurodegenerative disorders caused by a trinucleotide repeat expansion via in vivo genome editing
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hPSC-Derived Striatal Cells Generated Using a Scalable 3D Hydrogel Promote Recovery in a Huntington Disease Mouse Model.
Huntington disease (HD) is an inherited, progressive neurological disorder characterized by degenerating striatal medium spiny neurons (MSNs). One promising approach for treating HD is cell replacement therapy, where lost cells are replaced by MSN progenitors derived from human pluripotent stem cells (hPSCs). While there has been remarkable progress in generating hPSC-derived MSNs, current production methods rely on two-dimensional culture systems that can include poorly defined components, limit scalability, and yield differing preclinical results. To facilitate clinical translation, here, we generated striatal progenitors from hPSCs within a fully defined and scalable PNIPAAm-PEG three-dimensional (3D) hydrogel. Transplantation of 3D-derived striatal progenitors into a transgenic mouse model of HD slowed disease progression, improved motor coordination, and increased survival. In addition, the transplanted cells developed an MSN-like phenotype and formed synaptic connections with host cells. Our results illustrate the potential of scalable 3D biomaterials for generating striatal progenitors for HD cell therapy
A Rationally Designed, General Strategy for Membrane Orientation of Photoinduced Electron Transfer-Based Voltage-Sensitive Dyes
hPSC-Derived Striatal Cells Generated Using a Scalable 3D Hydrogel Promote Recovery in a Huntington Disease Mouse Model.
Huntington disease (HD) is an inherited, progressive neurological disorder characterized by degenerating striatal medium spiny neurons (MSNs). One promising approach for treating HD is cell replacement therapy, where lost cells are replaced by MSN progenitors derived from human pluripotent stem cells (hPSCs). While there has been remarkable progress in generating hPSC-derived MSNs, current production methods rely on two-dimensional culture systems that can include poorly defined components, limit scalability, and yield differing preclinical results. To facilitate clinical translation, here, we generated striatal progenitors from hPSCs within a fully defined and scalable PNIPAAm-PEG three-dimensional (3D) hydrogel. Transplantation of 3D-derived striatal progenitors into a transgenic mouse model of HD slowed disease progression, improved motor coordination, and increased survival. In addition, the transplanted cells developed an MSN-like phenotype and formed synaptic connections with host cells. Our results illustrate the potential of scalable 3D biomaterials for generating striatal progenitors for HD cell therapy
A Rationally Designed, General Strategy for Membrane Orientation of Photoinduced Electron Transfer-Based Voltage-Sensitive Dyes
Voltage
imaging with fluorescent dyes offers promise for interrogating
the complex roles of membrane potential in coordinating the activity
of neurons in the brain. Yet, low sensitivity often limits the broad
applicability of optical voltage indicators. In this paper, we use
molecular dynamics (MD) simulations to guide the design of new, ultrasensitive
fluorescent voltage indicators that use photoinduced electron transfer
(PeT) as a voltage-sensing switch. MD simulations predict an approximately
16% increase in voltage sensitivity resulting purely from improved
alignment of dye with the membrane. We confirm this theoretical finding
by synthesizing 9 new voltage-sensitive (VoltageFluor, or VF) dyes
and establishing that all of them display the expected improvement
of approximately 19%. This synergistic outworking of theory and experiment
enabled computational and theoretical estimation of VF dye orientation
in lipid bilayers and has yielded the most sensitive PeT-based VF
dye to date. We use this new voltage indicator to monitor voltage
spikes in neurons from rat hippocampus and human pluripotent-stem-cell-derived
dopaminergic neurons