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Biomaterial approaches to gene therapies for neurodegenerative disorders of the CNS

Biomaterial approaches to gene therapies for neurodegenerative disorders of the CNS
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  BiomaterialsScience REVIEW Cite this:  Biomater. Sci. , 2013,  1 , 556 Received 1st February 2013,Accepted 19th March 2013DOI: 10.1039/c3bm60030k Biomaterial approaches to gene therapies for neurodegenerative disorders of the CNS Ben Newland, a Eilís Dowd b and Abhay Pandit* a Neurodegeneration gives rise to a wide range of disorders which represent a growing health burden toboth western societies and developing countries. Whilst for many disorders such as Alzheimer ’ s andParkinson ’ s disease the cause is unknown, gene therapy is becoming the forefront of novel potentialtherapies described in the literature and has entered clinical trials. Furthermore, although in somewhatan earlier stage, biomaterials o ff er means of enhancing gene therapy strategies either through new deliv-ery methods or provision of support for genetically manipulated cells. This review outlines recent uses ofbiomaterials in the CNS and captures recent advances in non-viral gene delivery to the brain. Threedimensional sca ff olding systems for  ex vivo  gene delivery to the brain are also discussed highlighting theprogress of hydrogel mediated cell delivery. This review also addresses the di ffi culties and safety consider-ations of these approaches; illustrating the ability of biomaterial strategies to signi fi cantly improve out-comes of gene therapies for neurodegenerative disorders. 1 Introduction Neurodegenerative disorders such as Alzheimer ’ s disease (AD),Parkinson ’ s disease (PD), amyotrophic lateral sclerosis (ALS),Huntington ’ s disease (HD) and multiple sclerosis (MS) are allcharacterised by the loss of neuron or glial cells in the brain orspinal cord. These conditions are progressive in nature, unlikeother forms of neurodegeneration such as cerebrovascularaccidents (stroke), or those caused by external factors,  i.e. spinal cord injury (SCI) and traumatic brain injury (TBI).Dementia, of which AD is the leading cause, is the most common of the neurodegenerative disorders, with an esti-mated 24 million people being a ff  ected worldwide. 1 Thesecond most common neurodegenerative disorder, PD, has anincidence of 8.6 to 19.0 per 100000 inhabitants. 2 In contrast  Ben Newland  Ben Newland is currently pursu-ing a PhD in Professor Pandit  ’  slaboratory at the National Uni-versity of Ireland, Galway. He graduated with a BSc in Natural Sciences (chemistry/biology)  from Durham University (UK). He later graduated with an MResin Nanomaterials from Imperial College London (UK) beforebeginning his PhD research that  focuses on the use of biomater-ials to assist gene therapies for  Parkinson ’  s disease. Eilís Dowd  Dr Eilís Dowd is currently a Lec-turer in Pharmacology at  National University of Ireland,Galway (Ireland). She earned her  PhD at Edinburgh University(UK) after which she wasawarded a Wellcome Trust Tra-velling Postdoctoral Fellowshipwhich enabled her to complete post-doctoral research at McGill University (Canada) and Cardi   ff  University (UK). In 2006 she  joined the faculty at National University of Ireland, Galway. Her current research is focused on developing and validating novel  pharmacological, cell and gene therapies for neurodegenerativediseases. a  Network of Excellence for Functional Biomaterials (NFB), National University of  Ireland, Galway, Ireland. E-mail:; Fax: +353 91 495585;Tel: +353 91 492758 b  Pharmacology and Therapeutics, National University of Ireland, Galway, Ireland  556  |  Biomater. Sci. , 2013,  1 , 556 – 576 This journal is © The Royal Society of Chemistry 2013    P  u   b   l   i  s   h  e   d  o  n   0   5   A  p  r   i   l   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   0   1   /   1   1   /   2   0   1   3   1   3  :   5   6  :   0   2 . View Article Online View Journal | View Issue  to AD, where 60% of su ff  erers live in developing countries, PDis most prevalent in the USA and Europe. 1,3  Whilst others areless common, the majority of neurodegenerative disorders areage related diseases, so through the general rise in ageing demographics, these disorders are likely to become moreprevalent ( e.g.  a predicted 80 million will su ff  er from AD glob-ally by 2040). 1  Whilst the mechanisms involved in the pathogenesis of these diseases are in various states of elucidation, the aetiology for the majority of progressive cases remains unknown (withthe exception of those caused by genetic inheritance  e.g.  famil-ial PD 4 – 6 or HD 7 ). Another problem for patients of such dis-eases is that there are very few disease modifying therapies(such that exist are solely for the relapsing-remitting form of MS 8 ). Thus, although therapies such as oral levadopa for PDpatients o ff  er a substantial rise in the quality of life, thesetherapies do not retard neuronal cell death. Whilst this may paint a bleak picture of the current state of therapies for thetreatment of neurodegenerative diseases, one must also con-sider the inherent nature of the diseases that give a positiveoutlook for future interventions. The first aspect of thesedegenerative states is that the progression is often slow (withthe most notable exceptions being SCI, TBI, stroke and to alesser extent ALS), which gives a window of opportunity forintervention. The second is that neurons are influenced by growth factors such as nerve growth factor (NGF), 9 brain-derived neurotrophic factor (BDNF) 10 and glial-derived neuro-trophic factor (GDNF), 11 to name a few. The neuroprotectiveproperties of these trophic factors is well documented, 12 – 15 and they o ff  er an exciting potential for early intervention withthe more progressive diseases coupled with the possibility of tissue repair in the acute disorders.The first growth factor to be identified, NGF, 9  was found toprotect cholinergic neurons in adult rats following axotomy, 13 – 15 and the potential for therapeutic benefit in AD was noted that same year. 16 However, the size of proteins andtheir inherent charge renders them incapable of crossing theblood – brain-barrier (BBB). Therefore, as with all protein infu-sion trials, the protein must be delivered intracerebrally. Clini-cal trials based on the direct injection/infusion of growthfactors into the brains of patients with PD or AD, have resultedin varying outcomes. By a brief review of these trials one canhighlight some potential problems inherent with direct protein delivery. Amidst rising concerns over the safety of intracerebroventricular injections of NGF, 17,18 a clinical trialinvolving three patients with AD was undertaken, whereby thepatients received continuous intracerebroventricular infusionsfor a period of three months. 19 Due to the short half-life of growth factors, a continuous infusion directly into the lateral ventricle was required. However, this trial showed no improve-ment in cognitive tests and had to be discontinued due topatient weight loss and pain. Whilst these side e ff  ects hadbeen predicted in animal models, the lack of patient ameliora-tion may be contributed to the lack of labelled NGF localising in basal forebrain neurons of primates following similaradministrations. 20 These studies therefore suggest that direct administration to the target tissue was required. Nevertheless,it must be noted that one major advantage of growth factoradministrations is that the negative side e ff  ects reversed whendelivery ceased. 19  A more targeted approach to growth factor delivery wasreported in 2003 for five patients with PD who participated ina Phase I trial. Intraputaminal administration of GDNF, that acts upon dopaminergic neurons (neurons lost during PD), forthe course of a year produced an overall improvement in theUnified Parkinson ’ s Disease Rating Scale (UPDRS) score for allfive patients. 21  Another Phase I trial, involving ten patientsreceiving intraputaminal administration of GDNF, led tosimilar conclusions; that an overall benefit was observedduring the 12 months, with only minor side e ff  ects. 22 However the trial was halted by the sponsor Amgen following blood sample analysis detecting GDNF binding antibodies inseven of the ten patients. A follow up study of the patients one year post-treatment showed no overt adverse e ff  ect from this,but showed that the melioration due to GDNF was reversed without the continuation of treatment. 23 In contrast to thesetrials, where improvements in UPDRS scores were observed, arandomised, multicentre, double blind and placebo con-trolled trial involving thirty four PD patients showed no sig-nificant improvement in UPDRS score by intraputaminaladministration of GDNF. 24 However, much criticism of thestudy has arisen especially over delivery-specific issues suchas the type of cannula used (di ff  ering from the multiport oneused in the first study) and dose used (lower than both theprevious studies). 25 Though the protective e ff  ects of neuro-trophic factors have been well reported, it is likely that issuessurrounding optimised delivery remain a cause for a lack of overall e ffi cacy of these therapies. 26 In light of these findings, the potential benefits that genetherapy based approaches to neurodegenerative diseases may   Abhay Pandit   Prof. Abhay Pandit is the Direc-tor of a Science Foundation Ireland funded Strategic Research Cluster   “   Network of  Excellence for Functional Bioma-terials ”   (NFB) at the National University of Ireland, Galway. Prof Pandit  ’  s postgraduate work  focused on the modification of a fibrin sca  ff  old to deliver a thera- peutic biomolecule that resulted in a clinical trial at the BurnCentre at the University of  Alabama at Birmingham (USA). He worked in the medical device industry in the USA for seven years. He has been at the National University of Ireland, Galway for the last ten years. His current research interests include devel-oping responsive extracellular matrix-based systems for biomolecu-lar delivery for a range of clinical targets. Biomaterials Science Review This journal is © The Royal Society of Chemistry 2013  Biomater. Sci. , 2013,  1 , 556 – 576 |  557    P  u   b   l   i  s   h  e   d  o  n   0   5   A  p  r   i   l   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   0   1   /   1   1   /   2   0   1   3   1   3  :   5   6  :   0   2 . View Article Online  o ff  er must be assessed. By grouping gene therapies for brainapplications into two major subcategories  –  direct   in vivo  genedelivery and indirect   ex-vivo  gene therapy   –  it is noted that both approaches have the potential to mediate therapeutice ff  ects with a single, one-time administration. In the case of using these approaches to manipulate over-expression of neu-rotrophic proteins either in host cells ( in vivo ) or implantedcells ( ex vivo ), single administrations could be su ffi cient, pro- vided that suitable e ffi cacy can be achieved. Whilst reaching such levels of e ffi cacy remains a target (to which the use of bio-materials can be a useful aide  –  discussed later) the single sur-gical procedure will obviate the implantation of a catheter intothe patient  ’ s brain. In doing so, catheter related problemssuch as local excoriation, 23 infection, 21 migration from thedesired position 24 and the need for surgical revision for cath-eter re-positioning  21 can be eliminated. Another drawback of the direct infusion process needed for growth factor therapiesalso drives a positive rationale to turn focus towards genetherapies. Although this e ff  ect is less obvious, it arises inapplications where a large spread of the neurotrophic factor isrequired ( i.e.  in the relatively large structure of the striatum forPD) and may incur serious implications. In order to increasethis di ff  usion distance of the neurotrophic factor from thecannula port, convection enhanced delivery (CED) can beused. 27 The process involves the infusion of a relatively largeliquid volume (normally in pulses) that relies on the localpressure to drive the liquid through the brain interstitium.However, analysis of the brains of adult rhesus monkeys ten weeks post CED of GDNF showed gross tissue loss of up toapproximately 2 mm in diameter around the catheter tip. 28  Although the authors attribute this tissue damage to local flow rates from the single port catheter, potential methods of coun-tering this negative side e ff  ect are not discussed. It has beenfound that a single four micro litre injection of phosphatebu ff  ered saline into the striatum of adult Sprague Dawley ratsdoes not result in such gross tissue loss. 29 Thus, provided that acute toxicity of the  in vivo  or  ex vivo  approach can be avoided,the single injection for a gene therapy strategy is less detrimen-tal to the surrounding tissue. This review will therefore focuson an injectable, single dose therapeutic interventionmediated through the use of biomaterials. 2 Neurological disorder speci fi cbiomaterials toolbox Using biomaterials to mediate a one-o ff   intervention strategy has the foreseeable benefits as outlined in the introduction.Biomaterials can be used in several gene based strategies forneurological therapies. Potentially, such materials can there-fore be used to replace existing viral gene vectors, as controlleddelivery devices to the CNS, for neuron regeneration  via  func-tionalised sca ff  olds and as a method of enhancing   ex vivo  celldelivery. It is also highly feasible for a combination of thesefeatures to be used together in a complementary or combina-torial role, as will be described later on. Fig. 1 shows aschematic representation of these possibilities which are inrelative infancy for neural applications. However, much pro-gress has been made in areas such as non-viral gene delivery as a general shift can be noted from early non-targeted vectors(see Tables 1 and 2) to new nanoparticle formations (Table 3)and targeted vectors that to deliver genetic cargo to specifictissue or cell types (Table 4). 2.1 Non-viral gene vectors  An important research focus for materials design in genebased therapies for neurological disorders is in replacement of the existing viral vectors themselves. Whilst a number of safety concerns have arisen through the use of viral vectors, 30 – 33 several recent trials have shown the safety of non-genome inte-grating viral vectors. 34 However, for therapies to enter wide-spread use, they must be economically viable in terms of product manufacture, preparation procedures and adminis-tration techniques. Non-viral gene vectors often o ff  er the possi-bility of facile upscale and safe handling procedures, which,along with the ability to incorporate large plasmids, promisesubstantial benefits over their viral counterpart. The use of polymeric or liposomal gene vectors in the brain has thereforebeen the focus of much research.The majority of non-viral gene vectors use a charge – chargeinteraction to condense nucleic acids ( e.g.  DNA, siRNA, mRNA  etc. ) through the negative charge of the nucleic acids arising through the phosphate groups, and cationic moieties on the vector. This interaction, resulting in a particle commonly termed with the su ffi x   “ -plex  ” , for example polyplex (polymerformed complex), often condenses the DNA to such an extent that intercalating dyes can no longer interpolate with the DNA base pairs. 37 These charge based non-viral nucleic acid vectors, reviewed elsewhere, 38 have been prepared in a variety of forms outlined in Fig. 2, ranging from the conventionalpolymer or liposomal vectors 39 to the more recent nanoparticleor nanotube vectors. Of these vectors, polyethylenimine (PEI)based vectors 40 and liposomal vectors 41 have been the focus of much research attention and are discussed later in the context of use in the mammalian brain.The majority of research in the field of non-viral vectorsaims at increasing vector e ffi ciency whilst reducing its toxicity.Increasing e ffi ciency may be sought through: changing vectorcomposition, 42  varying the molecular weight of the vector, 43 altering vector structure, 44,45 mediating selective intracellulardegradation, 46 increasing circulation time (for systemically administered vectors), 47,48 membrane disruption 49 or increas-ing the specificity of targeting, 50 – 52 to name a few. Whilst thereis often a clear relationship between the vector to nucleic acidratio and the toxicity of the system as a whole ( e.g.  polyplex/lipoplex   etc. ) 53 the majority of work aimed at reducing toxicity sought to reduce the toxicity of the vector itself. Polyethyleneglycol (PEG), a neutral polyether, has been used extensively toalter the properties of biomedical materials through its intrin-sic properties of good solubility with a lack of toxic e ff  ects. 54 Modification of gene vectors with PEG has, to a large extent,focused upon reducing the vector toxicity, 55  which can shield Review Biomaterials Science 558  |  Biomater. Sci. , 2013,  1 , 556 – 576 This journal is © The Royal Society of Chemistry 2013    P  u   b   l   i  s   h  e   d  o  n   0   5   A  p  r   i   l   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   0   1   /   1   1   /   2   0   1   3   1   3  :   5   6  :   0   2 . View Article Online  surface charge 56 and reduce protein/blood component aggre-gation. 57  Another common use of PEG is as a spacer between vector and targeting moieties. However, when considering thecommercial feasibility of up-scaling non-viral vectors forapplications in neurological disorders, one must consider that the majority of PEGylation strategies involve post modificationof the srcinal vector, 58 – 61 i.e.  an additional synthesis/purifi-cation step. An attractive alternative lies in chemistries that  Fig. 1  Schematic representation of various biomaterial constructs that can aid gene therapy applications for neurodegenerative disorders: nucleic acid vectors (a),sustained delivery devices (b), neural conduits (c) and cell support constructs (d). Subsequent microscopy images show: transmission electron micrograph of poly-plexes formed by condensation of plasmid DNA with a polymeric vector (e), scanning electron micrograph of collagen hollow spheres prior to polyplex loading (f),scanning electron micrograph of PC12 cells (pseudocoloured) growing on an aligned collagen sca ff old (scale bar = 25  μ m) (g) and  fl uorescent micrograph of GFPtransgenic stem cells growing within a collagen hydrogel (h) taken with permissions 29,35,36 (e – h). Biomaterials Science Review This journal is © The Royal Society of Chemistry 2013  Biomater. Sci. , 2013,  1 , 556 – 576 |  559    P  u   b   l   i  s   h  e   d  o  n   0   5   A  p  r   i   l   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   0   1   /   1   1   /   2   0   1   3   1   3  :   5   6  :   0   2 . View Article Online  incorporate PEG during the srcinal synthesis producing aPEGylated vector after a single purification step, 62 allowing low cost possibilities for the scale-up of safe vectors that would berequired for such intervention strategies. 2.2 Controlled delivery devices In order to overcome the problem of the short half-life of growth factors, controlled delivery devices have been designedto function as a sustainable protein release platform, thusallowing an increased action time of the growth factor. 63,64 Synthetic polymers such as poly( D , L -lactide- co -glycolide) (PLGA)have been used to fabricate biodegradable microspheres that do not augment the host response to implantation in the rat brain. 65 Furthermore, microspheres based on such polymers,can be loaded with growth factors such as NGF, 66,67 GDNF 68 and NT-3 69 for sustained delivery applications. Other syntheticmaterials such as PEG have been used to create microspheresfor the fabrication of protein gradients, whereby di ff  erent den-sities of microspheres are graded during centrifugation. 70  Attractive alternative materials for the manufacture of con-trolled delivery devices are natural biopolymers such as fibrin,chitosan, collagen, elastin and hyaluronan. Recently, attentionhas been drawn towards the use of carriers based on thesematerials to deliver plasmid DNA, often in a complexed form Table 1  Liposomal transfection agents and nucleic acids delivered to the mammalian brain  Vector Gene Methods OutcomesResearcher/YearLipofectin®  β -Galactosidase orchloramphenicolacetyltransferaseDirect injection into the neonatalmouse brain, unspecified conditions,analysed up to 9 days post injection.Qualitative X-gal positive staining orchloramphenicol acetyltransferaseimmunopositive cells in several brain regions.Ono  et al  .,1990 130 Lipofectin®  β -Galactosidase orcholecystokininoctapeptideStereotactic ICV  a injection intoP77PMC rat model of audiogenicepileptic seizures, analysed up to 14days post injection. ∼ 3 fold reduction in the vulnerability of seizure score 4 days after injection.Li-Xin  et al  .,1992 131 Lipofectin®  β -Galactosidase Stereotactic injection into the caudateputamen of the adult mouse.Heterozygous deficient gus mps/+ mouse used to control for falsepositive X-gal staining. Analysis up to21 days post injection.Qualitative analysis of X-gal positive staining,found largely along needle tract, no obviousdi ff  erence between promoters used.Rossler  et al  .,1994 132 DOGS b /DOPE c Luciferase or β -galactosidaseDirect injection approximately intothe striatum of the newborn mouse. Analysis conducted 1, 2 and 3 dayspost injection.Quantitative analysis show highest luciferaseexpression with low charge ratios (0.8 and1.8), 1  μ g of DNA and 24 h post injection.Schwartz et al  ., 1995 133 Lipofectin®  β -Galactosidase orcholecystokininStereotactic ICV  a injection intoP77PMC rat model of audiogenicepileptic seizures. Also injection intothe hippocampus for visualisation of transfection. 1 – 21 day analysis.Successful suppression of audiogenic seizures4 days post injection (10  μ g/20  μ l). QualitativeX-gal positive staining of ventricularependymal cells.Zhang   et al  .,1997 135 Lipofectin®  β -Galactosidase Either single stereotactic injection orcontinual infusion with Alzet®osmotic minipumps in the adult rat septum. Animals were sacrificed 4and 6 days post surgery.Qualitative analysis. X-gal staining provednon-specific,  β -gal immunohistochemistry revealed only low intensity staining,concluding low e ffi ciency of transfection.Kofler  et al  .,1998 136 DOGS b β -Galactosidase Continual infusion with Alzet®osmotic minipumps in the adult rat caudate putamen. Analysis 1, 7 and 14days post injection.Quantification of transfected cells following  β -gal immunohistochemistry. Highest transfected cell number after 7 days of continual infusion or highest dose (50  μ g).Imaoka  et al  .,1998 137 DOGS b Tyrosine hydroxylase(TH) or L-AADC d  7 day continual infusion into thestriatum of the 6-OHDA  e rat model of PD. Functional recovery assessed by apomorphine induced rotations.Functional improvement through THtransfection up to 6 weeks post infusion.Improved further by dual infusion of both THand L-AADC.Imaoka  et al  .,1998 138 Lipofectin® TH Stereotactic injection into two sites of the striatum of the 6-OHDA  e rat model of PD.Functional improvement for up to 3 weeks asassessed by apomorphine induced rotations. Astrocyte specific expression of TH.Segovia  et al  .,1998 144 MLRI   f   -DOPE c Luciferase, GFP  g  or Hsp70 h Stereotactic ICV  a injection of vectorsinto the adult rat ventricle. Analysis 1day or 44 h post injection.Qualitative immunohistochemical analysisshow cells stained positive for Hsp70. GFPexpression detected by immunofluorescence.Hecker  et al. ,2001 153  JetSI ™ /DOPE c Luciferase siRNA Direct injection of siRNA formulations along with plasmidformulations into approximately the ventricular area of the newbornmouse. Analysis up to 60 h post injection.Maximal downregulation of the polymerinduced upregulation of luciferase expressionachieved at a dose of 0.05 pico moles of siRNA.Hassani et al. , 2005 150 a Intracerebroventricular.  b Dioctadecylamido glycylspermine.  c Dioleoylphosphatidyl ethanolamine.  d   Aromatic  L -amino acid decarboxylase. e 6-Hydroxydopamine.  f   Myristoyl lauroyl rosenthal inhibitor.  g  Green fluorescent protein.  h Heat shock protein. Review Biomaterials Science 560  |  Biomater. Sci. , 2013,  1 , 556 – 576 This journal is © The Royal Society of Chemistry 2013    P  u   b   l   i  s   h  e   d  o  n   0   5   A  p  r   i   l   2   0   1   3 .   D  o  w  n   l  o  a   d  e   d  o  n   0   1   /   1   1   /   2   0   1   3   1   3  :   5   6  :   0   2 . View Article Online
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