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siRNA Delivery Across the Blood Brain Barrier to treat Huntington's Disease
 
General overview sentence or two
TR Yellow Team: Hannah Kempton and Meryem Ok
 
 
==Overview: siRNA Delivery Across the Blood Brain Barrier to treat Huntington's Disease==
*This project proposal aims to optimize the delivery of nucleic acids, specifically siRNAs, across the blood brain barrier (BBB) using nanotechnology, with an overall goal of knocking down mutant protein (mHtt) in Huntington's disease


==Background==
==Background==
===Blood Brain Barrier===
===Blood Brain Barrier===
#The blood brain barrier (BBB) plays a crucial role in modulating cerebral homeostasis and directing neuronal functions. It separates circulating blood from cerebrospinal brain fluid and prevents harmful toxins from reaching the brain.
*The blood brain barrier (BBB) plays a crucial role in modulating cerebral homeostasis and directing neuronal functions. It separates circulating blood from cerebrospinal brain fluid and prevents harmful toxins from reaching the brain.
#In other parts of the body, the thin endothelial cells lining vessel and capillary walls overlap at leaky junctions that are flexible enough to allow larger molecules like hormones, viruses, and bacteria to squeeze through the junctions or diffuse directly through the cells into the surrounding tissue.  
*In other parts of the body, the thin endothelial cells lining vessel and capillary walls overlap at leaky junctions that are flexible enough to allow larger molecules like hormones, viruses, and bacteria to squeeze through the junctions or diffuse directly through the cells into the surrounding tissue.  
#However, the endothelial cells lining blood vessels in the brain overlap at tight junctions and are surrounded by a thick basal membrane containing contractile pericytes as well as astrocytic glial cells that provide nutrients and play a role in brain and spinal cord repair. These cells are collectively known as the BBB.
*However, the endothelial cells lining blood vessels in the brain overlap at tight junctions and are surrounded by a thick basal membrane containing contractile pericytes as well as astrocytic glial cells that provide nutrients and play a role in brain and spinal cord repair. These cells are collectively known as the BBB.
#The BBB only allows small, essential hydrophobic molecules like O2 and glucose and essential ions like Na+, K+, and Cl- to pass from the blood to the brain and central nervous system (CNS), but prevents the passage of molecules greater than about 500 Da.  
*The BBB only allows small, essential hydrophobic molecules like O2 and glucose and essential ions like Na+, K+, and Cl- to pass from the blood to the brain and central nervous system (CNS), but prevents the passage of molecules greater than about 500 Da.
 
[[Image:BBB.png|left|400px]]
Image from : http://www.daviddarling.info/encyclopedia/B/blood-brain_barrier.html
 
 
 
 
 
 
 
 
 


===Nanodelivery===  
===Nanodelivery===  
#Recently, the use of nanoparticles (NPs) has become increasingly common in solving environmental and medical problems, particularly in targeted drug delivery systems, among others
*Recently, the use of nanoparticles (NPs) has become increasingly common in solving environmental and medical problems, particularly in targeted drug delivery systems, among others
#Due to their size, NPs are unique in that their chemical, physical, and biological properties differ from that of their bulk materials, so they can be considered “new” material
*Due to their size, NPs are unique in that their chemical, physical, and biological properties differ from that of their bulk materials, so they can be considered “new” material
#Significance of Nanoscience in Medicine
*Significance of Nanoscience in Medicine
##Higher surface area:volume ratio → nanomaterials have higher reactivity, mechanical strength, and magnetic or electrical properties
**Higher surface area:volume ratio → nanomaterials have higher reactivity, mechanical strength, and magnetic or electrical properties
##NPs able to cross BBB and mediate repair of BBB damage that may be responsible for diseases like Alzheimer’s
**NPs able to cross BBB and mediate repair of BBB damage that may be responsible for diseases like Alzheimer’s
##For our purposes, NPs may serve as an effective vector, containing siRNA or other drugs, for targeted drug delivery across the BBB
**For our purposes, NPs may serve as an effective vector, containing siRNA or other drugs, for targeted drug delivery across the BBB
 
==Significance==
===Central Idea: Bridging the Gap===
*Combine established siRNA treatments that have been proven to be effective in knocking down specific genes using viral delivery with developed nanodelivery techniques in order to optimize a nanotechnology based delivery system to transport RNAi molecules across the blood brain barrier and into neurons
*Much of the work with RNAi in neurons has been done using viral delivery systems, like AAV
**but, cause immune response
**can also damage BBB
**also not currently accepted as therapeutic, and there may be mainstream reluctance and controversy of using viral delivery due to past failures (like accidentally giving people Leukemia!)
**provide potential for improvements in delivery due to recent developments in nanotechnology
*Will have many applications once the nanodelivery system is optimized: for this study we will focus on delivering siRNAs for the purpose of therapeutics in Huntington's Disease, but the optimized siRNA delivery technique will be widely applicable to treating/understanding various other brain pathologies and neuronal function
 
===Huntington's Disease===
*Autosomal dominant neurodegenerative disorder
*Caused by expansion of CAG repeat at 5' end of Htt gene
*Selected as pathology of focus for developing our RNAi delivery technique due to clear genetic cause that provides an obvious target for RNAi
**as mentioned before, work has been done to knock down the mutant Htt with RNAi but this was done using viral systems
*Also selected due to lack of effective current treatments
**the few drugs available have very damaging side effects
***http://www.mayoclinic.com/health/huntingtons-disease/DS00401/DSECTION=treatments-and-drugs
**due to the severity of the disease and the paucity of effective, specific treatments, Huntington's is a good disease to work with because any therapeutic developments could significantly improve treatment options for patients


==Experimental Overview==
===siRNA===
*Will deliver siRNA in order to knock down the expression of the mutant Htt protein (by stopping translation of the mHtt mRNA), which in the patient would alleviate disease symptoms
**siRNA has been used in the past because you can silence the mutant species without having to deal with the problems associated with trying to alter the genome itself
*siRNAs will be used from previous papers that demonstrated effective knockdown using these siRNAs with viral delivery (like in http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2008.05734.x/full )
**the focus of this study is not the siRNA design, but the nanodelivery method!


==Significance==
===in vitro BBB model===
The primary purpose of T7.2 is to construct a biological organism that is easier to model than the original biological isolate.  There are existing aspects of the natural isolate that make it particularly well suited to system-level analysis. We want to make every effort to preserve these aspects, which include:
*Optimization will be done in an'' in vitro'' model of the BBB, allowing rapid testing in various combinations/alterations of the nanoparticles
#Viability -- Here viability refers to the ability to propagate the new species for practical experimental purposes.
**this will allow us to focus on the optimization of the delivery without the time and complications (and $$) of starting with mouse models
#Existing knowledge of the functional genetic elements -- We must continue to harness the tremendous amount of knowledge gained from genetic and biochemical experiments of T7 over the past 60 years. To some extent, these studies are what allow our current models to as detailed as they are.
*Many ''in vitro'' models exist, one will be selected from this review http://onlinelibrary.wiley.com/store/10.1002/jps.23022/asset/23022_ftp.pdf?v=1&t=hopd7pgo&s=c216bb8635e117f544685247e32476cd3e98a62d once we have a better understanding of which one will be best for this study
#Relatively decoupled from host physiology -- T7 begins shutting off host transcription and solublizing the host genome within minutes of infection.  There are very few host proteins necessary for T7 infection. We do not want to purposely or incidentally increase the dependency of the phage upon the host so that we can minimize modeling the intricacies of host physiology.
 
#Coupling of entry and transcription -- This coupling leads to the natural organization of genes on the T7 genome. In addition, the timing of genome entry, to some extent, makes modeling the phage easier.  In addition, any importance of the ordering of elements on the natural isolate will be more relevant to T7.2.
===Main Experimental Goal: Optimization of nanodelivery method to successfully deliver siRNA across BBB===
====Phase 1====
*Optimize nanodelivery across BBB in model
**Current idea: use "trojan horse" Liposomes to cross via receptor-mediated transfer across the endothelial cells of the BBB
*** "The Trojan Horse Liposome Technology for Nonviral Gene Transfer across the Blood-Brain Barrier" http://www.hindawi.com/journals/jdd/2011/296151/#B19
**Things to optimize:
***Which receptor we try to get across (insulin or transferrin)
***Material liposome is made of
***Size
***Concentration
***Any coating
**This will just focus on how much of the siRNA gets across BBB
*Will quantify how much of the siRNA passes BBB (spectroscopy)
 
====Phase 2====
*Verify delivery of functional siRNA across BBB and into primary cells from Huntington patients using our optimized delivery
*siRNA will cross the artificial BBB, underneath which primary cells expressing the mutant Htt are growing
*The siRNA, after crossing the BBB, should enter the mHtt expressing cells via the same receptor-mediated transfer and knock down the mHtt mRNA
*This will be confirmed by harvesting the cells, extracting RNA, and doing RT-qPCR to quantify mHtt mRNA knockdown
 
===Future Work===
*Try actually doing it ''in vivo''
 
==References==
* Nanobiotechnology-Based Strategies for Crossing the Blood–Brain Barrier
**http://www.medscape.com/viewarticle/770396
**Intro
***Issues with some existing methods: damage bbb
***“ The ideal method for transporting drugs across the BBB should be controlled and should not damage the barrier. Among the various approaches that are available, nanobiotechnology-based delivery methods provide the best prospects for achieving this ideal. This review describes various nanoparticle (NP)-based methods used for drug delivery to the brain and the known underlying mechanisms.”
***“Some strategies require multifunctional NPs combining controlled passage across the BBB with targeted delivery of the therapeutic cargo to the intended site of action in the brain.”
***Fig 1 could be useful to base a figure off of in background info
***macromolecules can pass via receptor-mediated process (ex transferrin (Tf), insulin, immunoglobulin G)
***also remember some disorders increase BBB permeability as part of pathogenesis
***The upper limit of pore size in the BBB that enables passive flow of molecules across it is usually <1 nm; however, particles that have a diameter of several nanometers can also cross the BBB by carrier-mediated transport
**Ideal Method of Transport
***It should be controlled;
***It should not damage the barrier;
***The carrier system should be biodegradable and not toxic;
***Transport of drugs across the BBB should be selective;
***Systemic delivery should be targeted to the BBB and the site of intended action in the brain;
***The drug load transported through the BBB should be adequate for reaching therapeutic concentrations in the brain;
***Therapeutic concentrations should be maintained for a sufficient duration of time for the desired efficacy.
**NPs used
***Various factors that influence the transport include: type of polymer or surfactant used, NP size and the drug molecule.
***lipid NPs
***liposomes
***polymeric NPs (put Tf receptors on outside so can cross via Tf receptors) “molecular trojan horse”
****chitosan, dendrimers, nanogels, plga
***delivering siRNAs?
***lots on brain tumor treatment
 
*In vitro blood-brain barrier models: current and perspective technologies
**http://www.ncbi.nlm.nih.gov/pubmed/22213383
**“ In this article, we provide a detailed review and analysis of currently available in vitro BBB models ranging from static culture systems to the most advanced ow-based and three-dimensional coculture apparatus.”
**has good explanation for why in vitro models important: maybe reference in justifying why our study will be all in vitro
**discusses ideal characteristics of BBB model
***expression of tight junctions
***appropriate distribution of transporters
***efflux mechanisms (?)
***differentiating permeability
**for endothelial cells (ECs) to grow properly, need shear stress (flow) and cell-cell interactions (glia, astrocytes)
**describes many available models: we will select one of these that seems optimal (widely used, not too expensive)
 
*Gene Therapy for Huntington’s Disease
**http://www.ncbi.nlm.nih.gov/pubmed/22222669
**Adeno-associated viral (AAV) vectors
**Huntingtin (htt) gene discovered in 1993
***3000 aa, 350 kDa
***In normal individuals, 8 - 27 CAG (polyglutamine) repeats, increases to > 35 repeats in HD patients; cortical degradation also featured in pathology
***Motor phenomena → Chorea (random, spontaneous and involuntary dance-like movements), psychiatric symptoms (OCD, anxiety, paranoia, depression)
***Non-motor (cognitive) symptoms--executive functions, procedural memory, etc--often present years before other signs → more debilitating than motor symptoms
**“An ideal therapy for HD should not only target the triad of symptoms (motor, cognitive, psychiatric) experienced by patients but also halt the progression of these symptoms by preventing or stalling neuronal death. An additional challenge to any therapy is developing the best method of administration” → gene therapy
**2 main targets for preventing neuronal death in HD
***Growth factors provide cell w/needed nutritional/survival support → however, these typically work in animal models of CNS disorders but fail in clinical trials
***reducing mutant Huntingtin protein → Use RNAi
**Various classes of viral vectors
***adenovirus → problematic due to immunological issues
***lentivirus (LV) → retrovirus insert genetic material into host genome, used successfully in nonhuman primate models
***adeno-associated viral (AAV) → non-pathogenic, low immunogenicity, also used in animal models/clinical trials for neurodeg. disease, better at transducing certain brain areas compared to LV vectors
****Ideal because allow quick transduction (2- 4 weeks) of cells, sustained expression of gene of interest (GOI) for decades
***Problems with AAV (and viruses in general)
****different response in neonatal versus adult mice, possibly due to higher levels of astrocytes in adults (which trap AAV9)
****80% of population has antibodies to capsid proteins of WT AAV2/2, and 30 - 70% of these have antibodies that would neutralize capsid protein of AAV → prevents gene transfer in these patients, which would be a problem especially in repeated AAV exposure/administration


However, we feel the original biological isolate is not necessarily where we should begin to understand how system-behavior is produced. Specifically, in designing T7.2, the following five goals will drive our design; the first three goals revisit or extend goals motivating the design of [[T7.1]]. 
*Using non-coding small RNAs to develop therapies for Huntington's disease
#Our design of T7.2 will enable the unique and selection-independent manipulation of each genetic element via restriction enzymes.  
**http://www.nature.com/gt/journal/v18/n12/full/gt2011170a.html
#We will specify a genome that does not include any functions that might be encoded via the physical coupling of multiple genetic elements.
**As it is caused by a single, highly penetrant mutation at a defined locus, HD is potentially amenable to gene therapy using non-coding RNA.
#We will specify a genome that only includes elements that we believe contribute to phage gene expression.  Moving beyond our design of T7.1, we will actively erase or delete elements of unknown function.  In addition, efforts will be made to made to remove unknown genetic elements.
**a double-stranded RNA of 19–23 ribonucleotides directs gene silencing.
##reduced gene sets?
**It is known that most HD patients are heterozygous at the HD locus, carrying one mutant and one wild-type allele. Although mutant huntingtin protein is toxic to neurons, its wild-type counterpart is protective against apoptotic neuronal death. An effective gene therapy must silence the deleterious mutant allele without eliminating expression of the beneficial wild-type one.
##codon shuffling?
**Because of the lack of a transgenic animal model expressing both alleles of human wild-type and mutant HD gene, allele-specific silencing is conducted in cells from HD patients currently.
#To attempt to make our modeling of gene expression easier, we will use standard synthetic elements in place of the natural elements that regulate transcription and translation.
***i think we will definitely want to do ours in vitro, there seems to be a lot of iffy-ness about mouse models and i’m not sure that’s something we want to get into
#We will make a genome that is more anemenable to measurements that are important to us, such as adding reporters of transcription and translation.
**There are two sites in the mutant allele to which RNAi can target: mutant HD allele genetically linked SNP sites or expanded CAG repeats.
**goes into a lot of detail!!!


Taken together, our design of T7.2 should specify a genome that is simpler to model and manipulate, in which we have a putative function for each base pair of DNA involved in phage gene expression.  We hypothesize that as a result of the more parsimonious genome design, T7.2 will also encode a dynamic system that is easier to model and interact with, relative to the natural biological isolate.


==Meta Considerations==
*Self-assembling Modified β‑Cyclodextrin Nanoparticles as Neuronal siRNA Delivery Vectors: Focus on Huntington’s Disease
===Standardization===
**http://pubs.acs.org/doi/abs/10.1021/mp3003946
We want to standardize certain functional genetic elements to make them easier to model.  For example, instead of further characterizing every different ribosome binding site and promoter, we can standardize on a set of that would take considerably less effort to characterize.
**This paper is kind of similar to what we plan to do, but they only used their nanoparticles to get into neurons and did not account for BBB: They used a nanodelivery method that could deliver the siRNA across neuronal membranes, but then injected that directly into the brain, bypassing BBB completely
===Measurement===
**We can build off from that and say, well, this is a cool idea, but maybe it only works if you inject the NPs directly to the brain → ours should improve BBB passage so if effective it would be more therapeutically appealing (no injecting into patients brain)
We want to increase the ability to measure different aspects of phage biology.  This may including mRNA and protein reporters, and/or optimizing DpnI restriction sites to ease entry assays. 
===Manipulability===
We need to increase our ability to make selection-independent changes to the genome easily.  We will take the some of the lessons we learned in T7.1 about the types and distribution of restriction sites needed. 
===Encoding===
We want to reduce, to as large of an extent as possible, the number of genetic elements that are included in T7.2 that do not encode functions that we do not know about. 


==Designs==
*The Trojan Horse Liposome Technology for Nonviral Gene Transfer across the Blood-Brain Barrier
See the [[T7.2/Design|Designs Page]] for further information.
**http://www.hindawi.com/journals/jdd/2011/296151/#B19
**This is a method for delivering nucleic acids across BBB, we may optimize this technique for our delivery

Latest revision as of 17:59, 2 December 2013

TR Yellow Team: Hannah Kempton and Meryem Ok


Overview: siRNA Delivery Across the Blood Brain Barrier to treat Huntington's Disease

  • This project proposal aims to optimize the delivery of nucleic acids, specifically siRNAs, across the blood brain barrier (BBB) using nanotechnology, with an overall goal of knocking down mutant protein (mHtt) in Huntington's disease

Background

Blood Brain Barrier

  • The blood brain barrier (BBB) plays a crucial role in modulating cerebral homeostasis and directing neuronal functions. It separates circulating blood from cerebrospinal brain fluid and prevents harmful toxins from reaching the brain.
  • In other parts of the body, the thin endothelial cells lining vessel and capillary walls overlap at leaky junctions that are flexible enough to allow larger molecules like hormones, viruses, and bacteria to squeeze through the junctions or diffuse directly through the cells into the surrounding tissue.
  • However, the endothelial cells lining blood vessels in the brain overlap at tight junctions and are surrounded by a thick basal membrane containing contractile pericytes as well as astrocytic glial cells that provide nutrients and play a role in brain and spinal cord repair. These cells are collectively known as the BBB.
  • The BBB only allows small, essential hydrophobic molecules like O2 and glucose and essential ions like Na+, K+, and Cl- to pass from the blood to the brain and central nervous system (CNS), but prevents the passage of molecules greater than about 500 Da.

Image from : http://www.daviddarling.info/encyclopedia/B/blood-brain_barrier.html






Nanodelivery

  • Recently, the use of nanoparticles (NPs) has become increasingly common in solving environmental and medical problems, particularly in targeted drug delivery systems, among others
  • Due to their size, NPs are unique in that their chemical, physical, and biological properties differ from that of their bulk materials, so they can be considered “new” material
  • Significance of Nanoscience in Medicine
    • Higher surface area:volume ratio → nanomaterials have higher reactivity, mechanical strength, and magnetic or electrical properties
    • NPs able to cross BBB and mediate repair of BBB damage that may be responsible for diseases like Alzheimer’s
    • For our purposes, NPs may serve as an effective vector, containing siRNA or other drugs, for targeted drug delivery across the BBB

Significance

Central Idea: Bridging the Gap

  • Combine established siRNA treatments that have been proven to be effective in knocking down specific genes using viral delivery with developed nanodelivery techniques in order to optimize a nanotechnology based delivery system to transport RNAi molecules across the blood brain barrier and into neurons
  • Much of the work with RNAi in neurons has been done using viral delivery systems, like AAV
    • but, cause immune response
    • can also damage BBB
    • also not currently accepted as therapeutic, and there may be mainstream reluctance and controversy of using viral delivery due to past failures (like accidentally giving people Leukemia!)
    • provide potential for improvements in delivery due to recent developments in nanotechnology
  • Will have many applications once the nanodelivery system is optimized: for this study we will focus on delivering siRNAs for the purpose of therapeutics in Huntington's Disease, but the optimized siRNA delivery technique will be widely applicable to treating/understanding various other brain pathologies and neuronal function

Huntington's Disease

  • Autosomal dominant neurodegenerative disorder
  • Caused by expansion of CAG repeat at 5' end of Htt gene
  • Selected as pathology of focus for developing our RNAi delivery technique due to clear genetic cause that provides an obvious target for RNAi
    • as mentioned before, work has been done to knock down the mutant Htt with RNAi but this was done using viral systems
  • Also selected due to lack of effective current treatments

Experimental Overview

siRNA

  • Will deliver siRNA in order to knock down the expression of the mutant Htt protein (by stopping translation of the mHtt mRNA), which in the patient would alleviate disease symptoms
    • siRNA has been used in the past because you can silence the mutant species without having to deal with the problems associated with trying to alter the genome itself
  • siRNAs will be used from previous papers that demonstrated effective knockdown using these siRNAs with viral delivery (like in http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2008.05734.x/full )
    • the focus of this study is not the siRNA design, but the nanodelivery method!

in vitro BBB model

Main Experimental Goal: Optimization of nanodelivery method to successfully deliver siRNA across BBB

Phase 1

  • Optimize nanodelivery across BBB in model
    • Current idea: use "trojan horse" Liposomes to cross via receptor-mediated transfer across the endothelial cells of the BBB
    • Things to optimize:
      • Which receptor we try to get across (insulin or transferrin)
      • Material liposome is made of
      • Size
      • Concentration
      • Any coating
    • This will just focus on how much of the siRNA gets across BBB
  • Will quantify how much of the siRNA passes BBB (spectroscopy)

Phase 2

  • Verify delivery of functional siRNA across BBB and into primary cells from Huntington patients using our optimized delivery
  • siRNA will cross the artificial BBB, underneath which primary cells expressing the mutant Htt are growing
  • The siRNA, after crossing the BBB, should enter the mHtt expressing cells via the same receptor-mediated transfer and knock down the mHtt mRNA
  • This will be confirmed by harvesting the cells, extracting RNA, and doing RT-qPCR to quantify mHtt mRNA knockdown

Future Work

  • Try actually doing it in vivo

References

  • Nanobiotechnology-Based Strategies for Crossing the Blood–Brain Barrier
    • http://www.medscape.com/viewarticle/770396
    • Intro
      • Issues with some existing methods: damage bbb
      • “ The ideal method for transporting drugs across the BBB should be controlled and should not damage the barrier. Among the various approaches that are available, nanobiotechnology-based delivery methods provide the best prospects for achieving this ideal. This review describes various nanoparticle (NP)-based methods used for drug delivery to the brain and the known underlying mechanisms.”
      • “Some strategies require multifunctional NPs combining controlled passage across the BBB with targeted delivery of the therapeutic cargo to the intended site of action in the brain.”
      • Fig 1 could be useful to base a figure off of in background info
      • macromolecules can pass via receptor-mediated process (ex transferrin (Tf), insulin, immunoglobulin G)
      • also remember some disorders increase BBB permeability as part of pathogenesis
      • The upper limit of pore size in the BBB that enables passive flow of molecules across it is usually <1 nm; however, particles that have a diameter of several nanometers can also cross the BBB by carrier-mediated transport
    • Ideal Method of Transport
      • It should be controlled;
      • It should not damage the barrier;
      • The carrier system should be biodegradable and not toxic;
      • Transport of drugs across the BBB should be selective;
      • Systemic delivery should be targeted to the BBB and the site of intended action in the brain;
      • The drug load transported through the BBB should be adequate for reaching therapeutic concentrations in the brain;
      • Therapeutic concentrations should be maintained for a sufficient duration of time for the desired efficacy.
    • NPs used
      • Various factors that influence the transport include: type of polymer or surfactant used, NP size and the drug molecule.
      • lipid NPs
      • liposomes
      • polymeric NPs (put Tf receptors on outside so can cross via Tf receptors) “molecular trojan horse”
        • chitosan, dendrimers, nanogels, plga
      • delivering siRNAs?
      • lots on brain tumor treatment
  • In vitro blood-brain barrier models: current and perspective technologies
    • http://www.ncbi.nlm.nih.gov/pubmed/22213383
    • “ In this article, we provide a detailed review and analysis of currently available in vitro BBB models ranging from static culture systems to the most advanced ow-based and three-dimensional coculture apparatus.”
    • has good explanation for why in vitro models important: maybe reference in justifying why our study will be all in vitro
    • discusses ideal characteristics of BBB model
      • expression of tight junctions
      • appropriate distribution of transporters
      • efflux mechanisms (?)
      • differentiating permeability
    • for endothelial cells (ECs) to grow properly, need shear stress (flow) and cell-cell interactions (glia, astrocytes)
    • describes many available models: we will select one of these that seems optimal (widely used, not too expensive)
  • Gene Therapy for Huntington’s Disease
    • http://www.ncbi.nlm.nih.gov/pubmed/22222669
    • Adeno-associated viral (AAV) vectors
    • Huntingtin (htt) gene discovered in 1993
      • 3000 aa, 350 kDa
      • In normal individuals, 8 - 27 CAG (polyglutamine) repeats, increases to > 35 repeats in HD patients; cortical degradation also featured in pathology
      • Motor phenomena → Chorea (random, spontaneous and involuntary dance-like movements), psychiatric symptoms (OCD, anxiety, paranoia, depression)
      • Non-motor (cognitive) symptoms--executive functions, procedural memory, etc--often present years before other signs → more debilitating than motor symptoms
    • “An ideal therapy for HD should not only target the triad of symptoms (motor, cognitive, psychiatric) experienced by patients but also halt the progression of these symptoms by preventing or stalling neuronal death. An additional challenge to any therapy is developing the best method of administration” → gene therapy
    • 2 main targets for preventing neuronal death in HD
      • Growth factors provide cell w/needed nutritional/survival support → however, these typically work in animal models of CNS disorders but fail in clinical trials
      • reducing mutant Huntingtin protein → Use RNAi
    • Various classes of viral vectors
      • adenovirus → problematic due to immunological issues
      • lentivirus (LV) → retrovirus insert genetic material into host genome, used successfully in nonhuman primate models
      • adeno-associated viral (AAV) → non-pathogenic, low immunogenicity, also used in animal models/clinical trials for neurodeg. disease, better at transducing certain brain areas compared to LV vectors
        • Ideal because allow quick transduction (2- 4 weeks) of cells, sustained expression of gene of interest (GOI) for decades
      • Problems with AAV (and viruses in general)
        • different response in neonatal versus adult mice, possibly due to higher levels of astrocytes in adults (which trap AAV9)
        • 80% of population has antibodies to capsid proteins of WT AAV2/2, and 30 - 70% of these have antibodies that would neutralize capsid protein of AAV → prevents gene transfer in these patients, which would be a problem especially in repeated AAV exposure/administration
  • Using non-coding small RNAs to develop therapies for Huntington's disease
    • http://www.nature.com/gt/journal/v18/n12/full/gt2011170a.html
    • As it is caused by a single, highly penetrant mutation at a defined locus, HD is potentially amenable to gene therapy using non-coding RNA.
    • a double-stranded RNA of 19–23 ribonucleotides directs gene silencing.
    • It is known that most HD patients are heterozygous at the HD locus, carrying one mutant and one wild-type allele. Although mutant huntingtin protein is toxic to neurons, its wild-type counterpart is protective against apoptotic neuronal death. An effective gene therapy must silence the deleterious mutant allele without eliminating expression of the beneficial wild-type one.
    • Because of the lack of a transgenic animal model expressing both alleles of human wild-type and mutant HD gene, allele-specific silencing is conducted in cells from HD patients currently.
      • i think we will definitely want to do ours in vitro, there seems to be a lot of iffy-ness about mouse models and i’m not sure that’s something we want to get into
    • There are two sites in the mutant allele to which RNAi can target: mutant HD allele genetically linked SNP sites or expanded CAG repeats.
    • goes into a lot of detail!!!


  • Self-assembling Modified β‑Cyclodextrin Nanoparticles as Neuronal siRNA Delivery Vectors: Focus on Huntington’s Disease
    • http://pubs.acs.org/doi/abs/10.1021/mp3003946
    • This paper is kind of similar to what we plan to do, but they only used their nanoparticles to get into neurons and did not account for BBB: They used a nanodelivery method that could deliver the siRNA across neuronal membranes, but then injected that directly into the brain, bypassing BBB completely
    • We can build off from that and say, well, this is a cool idea, but maybe it only works if you inject the NPs directly to the brain → ours should improve BBB passage so if effective it would be more therapeutically appealing (no injecting into patients brain)
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