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DNA nanoparticle gene therapy in brain

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Dr David Yurek at the University of Kentucky, and his collaborator Dr Mark Cooper, a founder of Copernicus Therapeutics, Inc, are taking a novel approach to gene therapies in treating disorders of the brain. While the treatments for diseases such as Parkinson’s have developed, they have limited effectiveness and do not improve Parkinson’s natural history. This proficient team are applying their findings to discover innovative ways to overcome these limitations and improve Parkinson’s disease treatment.
Brain disorders are some of the most abundant, diverse and troublesome to treat physiological conditions clinicians encounter. Due to the complexity and inaccessibility of the brain, many of its associated disorders have suboptimal therapeutic options. Nonetheless, approaches such as gene therapy provide potential in overcoming these barriers.
Plasmid map of pGDNF_1b. This is the plasmid that was compacted into nanoparticles for most of the studies.
Gene therapies directly target depleted levels or mutant forms of proteins within cells, often using parts of DNA known as ‘gene segments’ incorporated into viral vectors. However, in the case of neurons, delivering and expressing the normal gene is challenging because neurons are post-mitotic (they do not reproduce), and there is a danger of viral vector immune responses.
Breaking through – nanoparticles
Alongside Dr Mark Cooper of Copernicus Therapeutics, Inc., Dr Yurek has used non-viral gene therapy vectors developed at Copernicus to treat neurological diseases, including Parkinson’s. These vectors are compacted into rod-like DNA nanoparticles (DNPs), and with these Dr Yurek and his research team were able to express the transfected gene within neurons and astrocytes to levels one hundred times that of naked plasmid DNA. It is the small size (8–11 nm) of these DNPs and their stability in nuclease-rich environments that allow them to diffuse safely and efficiently to the nucleus through the nuclear membrane envelope – an essential process for gene therapy of post-mitotic cells. Studies have demonstrated localisation of transfected gene expression to injection sites, and have revealed stable protein levels up to one year after a single treatment.

Due to the complexity and inaccessibility of the brain, many of its associated disorders have suboptimal therapeutic optionsQuote_brain

Furthermore, DNPs have been demonstrated to exhibit much fewer inflammatory, toxicity and immune responses when compared to viral vector approaches, strengthening promise for their clinical application. Ultimately, Dr Yurek, Dr Cooper and researchers at Copernicus Therapeutics hope to develop this approach as a treatment for Parkinson’s disease (PD) and other degenerative brain disorders.
Uses and improvements
During Dr Yurek’s and Dr Cooper’s studies investigating DNP performance and viability, the gene coding for glial cell line-derived neurotrophic factor (GDNF) was successfully transfected to cells of the rat striatum to produce overexpression of the protein – measured at up to six months post-injection. Interestingly, GDNF has been shown to restore dopamine loss in animal models of PD, and is also extremely beneficial to the success of embryonic dopaminergic cell grafts – another promising PD treatment.
Developing this, and the use of DNPs, Dr Yurek and collaborators at Copernicus Therapeutics have investigated the benefits of applying GDNF DNPs one week before introducing striatal dopaminergic grafts to rats. These studies discovered an increased survival and function of transplanted dopaminergic cells in comparison to GDNF DNP absence in the transplanted treated area. One graft pre-treated with GDNF DNPs was reported as 14 times larger than control mean graft size in absence of GDNF DNPs. Furthermore, PD behavioural characteristics in these animal models were lessened in conditions whereby GDNF DNP treatment preceded dopaminergic cell transplants. Together, these results show promise for improved embryonic neuronal cell grafts, offering an insight into one option for effective therapy of PD.

Alongside Dr Mark Cooper of Copernicus Therapeutics, Inc., Dr Yurek has
used non-viral gene therapy vectors developed at Copernicus to treat
neurological diseases, including Parkinson’sQuote_brain

Transmission electron micrograph of rod-like compacted nanoparticles containing the 4,064 bp GDNF_1b plasmid; nanoparticles were suspended in saline. The shape and size of the GDNF_1b DNPs are dependent on the length of the plasmid and compaction formulation, which included an acetate counterion. Scale bar = 100 nm; magnification = 40,000X.
Following the exciting outcomes of their investigations, Dr Yurek and Dr Cooper looked at optimising GDNF DNP transgenic activity and expression. From this they found that the shorter, more commonly used RNA splice variant gave rise to reduced GDNF secretion in comparison to its longer counterpart. Not only that, but synthetic DNA featuring codon optimisations (a method to increase the expression of a particular gene) also did not have a significant effect on gene activity or expression. They also noted that viral approaches to gene delivery can produce significantly more GDNF than DNP approaches. However, studies into effective levels of GDNF protein indicate that the amount DNPs increase GDNF expression is sufficient and, indeed, extremely high levels of over-expression could have adverse effects. Using DNPs was also found to be a much safer method, in comparison to viral methods. In reality, both approaches have characteristics that complement each other in treating disorders such as PD.
Exploiting benefits of injury
One particularly interesting finding from these studies into GDNF DNPs was the observation that DNPs elicit higher GDNF expression in the denervated striatum in comparison to an intact brain – in other words, they have a bigger effect on a neuronally damaged brain. This trend was also witnessed when comparing ages of treated animals – older, aged brains responded to GDNF DNPs with higher levels of expression than that of younger brains. Due to this pattern, Dr Yurek hypothesised that this effect was associated with the process of gliosis – the proliferation of astrocytes which increases with age and neuronal injury. This phenomenon and its foundations were explored, revealing, as expected, higher degrees of GDNF DNP activity and expression in denervated and older striatum.
Further study using either an astrocyte specific (GFAP) or non-discriminatory promoter revealed that gene activity in astrocytes impacted overall expression, but as shown previously, DNPs do transfect multiple types of brain cells – so other factors must be at play in this situation. Although this detail cannot be inferred here, findings from this particular study show great promise for DNP GDNF-based therapeutics. Here, neurodegeneration provides a foundation for DNP gene therapies to be extremely effective in this condition, as they can preferentially act on tissues that require regeneration.
The future’s bright!
The studies outlined above have made it possible to explore the use of compacted DNA nanoparticles. Dr Yurek’s and Dr Cooper’s work has provided great insights into nanoparticle function, therapeutic gene expression optimisation, neurodegenerative mechanisms and potential characteristics to focus on in designing successful treatments.
Following the success of their work, Dr Yurek and his collaborators at Copernicus Therapeutics aim to investigate the neuroprotective and restorative properties in dopaminergic neurons of GDNF DNPs, developing this line of treatment for PD. It is now hoped that these data can be transferred from animal models into human studies, and eventually clinical trials.
What made you realise the potential of improving genetic therapies for brain disorders?
Previous successful studies that used viral vectors as a means to transduce brain cells and overexpress therapeutic proteins provided the impetus to find an alternative and safer means to introduce therapeutic genes into brain cells. These nanoparticles have been found to be non-toxic when delivered to the lung of humans and when delivered to the nasal mucosa, lung, eye, and brain of animals.
Do you believe that DNP transfection alone can bring about marked improvements in Parkinson’s disease, enough for clinical use?
Nanoparticles may provide a means to deliver therapeutic genes to brain cells at physiologically relevant levels. This may be very significant when one considers that high expression of therapeutic proteins can lead to adverse off-target effects and can lead to the generation of antibodies that subsequently diminish the effects of these therapeutic proteins. In terms of clinical efficacy, “bigger is not always better” and therefore low, sustained expression of therapeutic genes may provide better long-term outcomes.
Have you looked into the specifics of treating any other neurological disorder with such an approach? What is the potential?
We have demonstrated these nanoparticles target both neurons and astrocytes, which suggests that other neurodegenerative disorders such as Alzheimer’s disease or traumatic brain injury may benefit from nanoparticle gene therapy. One of the cell surface receptors for nanoparticles, nucleolin, is highly expressed on the cell surface of some types of gliomas and therefore there may be some utility in using these nanoparticles to target brain tumours.
From these studies and your experience, can you see it being a smooth transition to DNP gene therapies in human cells?
One of the challenges of translating animal studies to human studies is the ability to scale-up the delivery of the nanoparticles to affect the larger brain structures in humans. These challenges can be overcome by using multiple deliveries of nanoparticles into specific brain regions or by developing a means for these nanoparticles to pass through the blood brain barrier at specific brain regions. Early data suggest this is possible using intranasal administration of DNA NPs.
How will your findings contribute to developing non-clinical data, such as research techniques and methodologies?
The use of gene therapy to express or knock-down (using RNA interference technologies) genes of interest may prove useful in exploring and understanding numerous biological pathways important for neurological processes, including neural outgrowth and connectivity, myelination, cell–cell recognition, formation of neural synaptic units and synaptic plasticity.
Research Objectives
Dr Yurek works in collaboration with Dr Mark Cooper and Copernicus Therapeutics, Inc. to research the functions and efficacy of using nanoparticles as a gene therapy technique for a number of neurological disorders.

  • National Institutes of Health (NIH)
  • National Institute of Neurological Disorders and Stroke (NINDS)
  • Michael J Fox Foundation for Parkinson’s Research

Dr Yurek received his Doctor of Philosophy degree in Physiology and Biophysics in 1987 from the University of Southern California. He completed his postdoctoral studies at the University of Rochester and is currently a Research Professor in the Department of Neurosurgery and a faculty member of the Nanobiotechnology Center at the University of Kentucky.
Dr Cooper received his BA degree from Cornell University and his MD degree from Johns Hopkins University. He joined the faculty at Case Western Reserve University School of Medicine where he was an Associate Professor of Medicine, is a founding scientist of Copernicus Therapeutics, and is now Senior VP of Science and Medical Affairs at Copernicus.
David Yurek, PhD (Professor)
Department of Neurosurgery
University of Kentucky College of Medicine
University of Kentucky Nanobiotechnology Center
Health Sciences Research Building, Room 230 1095 Veterans Drive Lexington
KY 40536-0305
T: +1 (859) 257-8219
E: [email protected]

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