Gene therapy 2010

Gene therapy    Definition: delivery and expression of genetic  material leading to an alteration in the instruction  set of a cell for a therapeutic purpose    1960’s ‐ idea of gene therapy proposed    1990 ‐ first human gene therapy clinical trial     2010 ‐ still no gene therapy product commercially  available     Somatic vs germ‐line gene therapy  Gene therapy ‐ making it work  What genetic material to deliver?Where to deliver it? How to deliver it?How to control it? How to test it? 1

Key goals for a gene therapy strategy    Easy to administer.     Long‐term production of a therapeutic protein or  therapeutic effect following a single application.    Highly specific, targeted to disease cells/tissue only,  thereby little or no side‐effects.  What genetic material to deliver?    Genes and RNA interference sequences    Correcting faults by:    Replacing a non‐functional gene with a functional copy    Silencing an abnormally functioning gene    Altering the phenotype of a cell    Need to know something about the disease process    Inherited or acquired, acute or chronic    Treatment outcome    Cure, alter natural history, alleviate symptoms  2

Where to delivery it?  During & Tipene-Hook, New Ethicals 1997 May, 57-65 Factors to consider   Many diseases affect multiple sites   Effects on circuitry e.g brain   Gene may need to be delivered to widespread areas  or restricted to a specific organ/group of cells  3

Getting genes into cells: gene delivery   Ex vivo ‐ gene transfer performed  in cell culture before  transplantation   In vivo ‐ gene transfer performed  in situ   Therapeutic gene ‐ transgene   Success of gene therapy  dependent on efficient gene  transfer    Gene transfer mediated by gene  delivery vehicles (=vectors)  www.biochem.arizona.edu/. ../Lecture25.html Gene delivery‐ non‐viral vectors    Plasmid DNA    Liposomes     DNA mixed with cationic lipids and polymers  (polylysine, protamine) becomes encased  within a lipid bubble.     Cellular uptake via an endocytic process.    No restriction on size of gene, easy and  cheap to manufacture.    Main disadvantages are relative inefficiency  in gene delivery and transient expression  4

Gene delivery ‐ viral vectors   Exploits natural ability of viruses to infect  mammalian cells   Genetically engineered to carry a transgene  cassette and deposit it within a cell.   Engineered for a single round of host cell infection  by removal of viral genes involved in replication.  VIRUS VIRAL PROTEINS ASSEMBLY SPREAD OF INFECTION VIRAL GENES 5

Life cycle of a viral vector  VECTOR GENE OF INTEREST DISPLACED VIRAL GENES PROTEIN OF INTEREST NO VIRAL GENES NO NEW VIRUS OR VIRAL PROTEINSEngineering vectors from viruses    Molecular cloning techniques used to genetically  engineer a virus to carry a transgene cassette    e.g. AAV rep and cap genes are removed and  replaced with a transgene gene cassette    Therapeutic genes are controlled by promoters,  regulatory elements    Cell lines are used to package vector particles   6

AAV vector transgene cassette      e.g AAV expression cassette ITR rep cap ITR Wild-type AAV ITR transgene expression cassette ITR Recombinant AAV 145 1100 <1600 600 300 145bp ITR promoter transgene RE pA ITR ITR – AAV inverted terminal repeat RE – regulatory element, e.g. woodchuck postregulatory transcriptional element (WPRE) pA – polyA signal Viral vectors ‐ types    Retrovirus    Herpes simplex virus    Adenovirus    Adeno‐associated virus (AAV)  7

  Retrovirus    7‐11kb ss RNA; Following target cell entry, RNA genome is reverse  transcribed into ds DNA which integrates into the cell chromatin    Efficient integration of transgene into chromosome leading to stable  gene expression which persists in parent and daughter cells.    Good for ex vivo approaches.    Main disadvantage ‐ potential for random integration near an  oncogene. Also unable to infect non‐dividing cells as it requires one  mitotic division for integration and expression of the transgene.     Lentivirus (HIV) ‐ subclass of retroviruses, infects both dividing and  non‐dividing cells, stable and long‐term gene expression.     Adenovirus    36kb dsDNA    Transduces non‐dividing and dividing cells    High titer vector produced    Main disadvantage ‐ transient transgene expression  due to immune responses directed against  adenoviral proteins; may be overcome by producing  “gutless” adenoviral vectors  8

  Herpes simplex virus (HSV1)    152kb linear ds DNA    Broad host cell range    Main disadvantage ‐ intrinsic toxicity and contamination of vector  stocks with wild‐type (pathogenic) virus. Difficulties in maintaining  long‐term gene expression   Adeno‐associated virus (AAV)    4.7kb ss DNA    >40 serotypes isolated from human, primates    Non‐pathogenic    Transduces non‐dividing and dividing cells     Long‐term gene expression (>2.5 yrs)    Main disadvantage ‐ small size  How do you choose the right vector for  the job?     Dependent on cell targets     Dividing vs non‐dividing cells, e.g stem cells and  post‐mitotic cells difficult to transduce, rapidly  dividing epithelial and cancer cells easy to transduce     Transduction = vector‐mediated gene transfer    Vector tropism = selectivity for a cell type    Dependent on the ligand present on a vector and  whether the target cell expresses the appropriate  cell‐surface receptor    Gene insert size  9

Achieving specificity ‐ vector targeting   Needed for:    Safety – minimises potential toxicity of gene product in healthy cells    Increases gene transfer efficiency – want to minimise amounts of  vector needed to produce gene product at therapeutic levels   Achieved by:    Mode of delivery e.g. direct injection into site of interest    Vector selection    Exploit or modify specificity of viral vectors for certain cell types  e.g AAV serotypes (human, primate)    Pseudotype vectors    Modify capsid shell that allows binding to different receptors  AAV2 AAV1/2eGFP expression in the hippocampus brain region following AAV2vector or AAV1/2 vector-mediated gene transfer How to control gene expression?    Why is it necessary?  Figure 2. Transduction of the target cell.The vector particle containing the therapeutic gene sequences binds to a cell, generally through a receptor-mediated process and then enters the cell, allowing the genome to enter the nucleus. The vector genome may go through complex processes but ends up as dsDNA that, depending on the vector, can persist as an episome or become integrated into the host genome. Expression of the therapeutic gene follows. Kay et al. 2001. Nat. Med., 7, 33-40. 10

Gene expression  Recombinant protein  Level of transgene expression  determined by promoter choice  and inclusion of other cis‐acting  DNA elements.   Aim is to achieve stable, long‐ term transgene expression.  Gene therapy Regulating transgene expression  Gene therapy  Essential in maintaining  transgene protein expression at  steady therapeutic levels   Enables flexibility in adjusting  dosage as disease evolves or for  therapies tailor‐made for  individual patients   Built in safety mechanism  Gene therapy - regulated Clackson, Gene Therapy, 7, 120-125, 2000. 11

Regulatory systems   Endogenous promoter‐regulated transcription systems that are responsive to  physiological stimuli    e.g. glucose responsive element ‐ control insulin release   Exogenous drug‐regulated transcription system    e.g. Rapamycin, ecdysone, tet system   Ideal regulatory system    Low basal expression    Be inducible to high levels over a wide dose range to provide a useful dose  responsiveness    Induction should be a positive effect ‐ i.e administer drug to switch it on    Drug should be active following oral administration and have no pleiotropic  effects in mammalian cells    Regulatory protein should have no effects on endogenous gene expression  and be of human origin to minimise immunogenicity  Tet On/Off regulatory system    Transactivator ‐ regulatory  element  – expresses the  tetracycline‐controlled  transactivator (rtTA) which is  a chimeric protein composed  of the Tet repressor fused to  the VP16 activation domain  of HSV.    Tet‐responsive element  (TRE) is upstream of a silent  promoter driving the  transgene of interest.  Adapted from: http://www.bdbiosciences.com/clontech/tet/index.html 12

How to test a gene therapy strategy?    Test functionality of therapeutic gene cassettes     In vitro and in vivo animal models of disease.    How adequate are those models and do they reflect  disease processes in humans?     Need measurable endpoints to assess whether  gene therapy has therapeutic effect and benefit    Assays for quantifying and/or visualising  therapeutic protein levels  Current status of gene therapy    Toxicity of some viral vector systems    Immune responses directed against cells containing foreign  proteins (viral) leading to elimination of the therapy. Thus the  therapy may be short‐lived.    Enhanced immune responses against vectors encountered  previously may mean problems in re‐dosing.    Potential for some vectors to recover ability to cause disease.    Vector targeting not optimised – transgenes expressed in  healthy and diseased cells    Safety mechanisms/control of gene expression not sufficiently  optimised and long‐term effects unknown    Multigene disorders    Best candidates for gene therapy are those that arise from  single gene mutations. Many common diseases (e.g Alzheimer’s,  heart disease) involve effects on a variety of genes.  13

Viral vectors: tools for gene function  studies   Gene overexpression    Express poorly characterised genes to gain a better  understanding of their physiological effects    Generating animal models of disease e.g.Parkinson’s  Huntington’s, Alzheimer’s diseases   Gene knockout     RNA interference  Advantages over transgenic animals   Can be engineered to express single or two foreign genes, different  regulatory elements and promoters   Can be administered at any developmental stage ‐ from in utero to adult  to senescent animals   Either short or long‐term CNS gene expression   Expression can be obtained in crucial brain regions while possible side‐ effects associated with widespread overexpression of the gene can be  avoided   Not host‐specific ‐ rats, primates, mice   Inexpensive and rapid to generate compared with classical transgenics   Higher levels of transgene expression obtained  1

Combined use of germline and viral  transgenic methods    Can be combined with transgenic mouse models for  studies of interactions of disease‐causing proteins  with other cellular proteins    Confirm gene specificity with transgenic knockouts.  Use viral vector‐mediated gene transfer of the  missing gene to rescue the phenotype    Observe further potentiation or downregulation of  gene effects    Affect regulation of genes in different tissues or  brain regions Rat and primate models of Parkinson’s  disease    AAV vectors used to overexpress wild‐type and  mutant forms of α‐synuclein in the substantia nigra  of rats and primates    Show many of the pathological features of PD   •  protein inclusions   •  dystrophic neurites  •  progressive loss of TH cells in the SNpc  •  drug‐induced rotation and motor deficits  2

Kirik D & Bjorklund A, Trends Neurosci. 26(7) 2003, 386-392 RNA interference (RNAi)    A form of post‐transcriptional gene silencing in which specific sequences  of double‐stranded RNA (dsRNA) can be used to knock down the  expression of a gene target    Adapted as a tool to investigate gene function    History of RNAi http://www.invitrogen.com/content.cfm?pageid=10088 3

Biochemical mechanism of RNAi  1.  dsRNA is introduced into the cell  2.  DICER digests dsRNA into ~21bp  dsDNA (short‐interfering RNAs;  siRNAs)  3.  The siRNAs are integrated into  the RNA Induced Silencing  Complex (RISC)  4.  siRNAs undergo strand  separation. The antisense strand  binds to its complementary/ target mRNA  5.  Argonaute ‐ endonuclease  within the RISC degrades the http://www.ambion.com/techlib/tn/101/7.html targeted mRNA  Using RNAi as a tool for manipulating  gene expression in mammalian cells    In mammalian cells, introduction of long dsRNA initiates  a cellular interferon response that ultimately causes cell  shutdown and leads to apoptosis    RNAi can be achieved in mammalian cells by direct  delivery of:    synthetic short‐interfering RNA (siRNA)   synthetic short‐hairpin RNA (shRNA)    microRNA (miRNA)  4

siRNA    21‐23 bp of double‐stranded RNA with a 3’ dinucleotide overhang    Algorithms are used to design RNAi sequences (guidelines provided e.g  30‐50% G/C content).     3‐5 siRNA sequences covering the gene of interest are selected and tested  for the degree of suppression of gene expression.  siRNA ‐ synthesis, delivery, effect    Synthesis in vitro    Chemical synthesis    In vitro transcription systems   Delivery    Cell culture ‐ transfection, electroporation    In vivo models ‐ injection or direct  application    RNAi effect ‐ transient (3‐7 days), partial to  full knockdown of gene expression    Davidson BL, Paulson HL. Lancet Neurol. 2004 Mar;3(3):145-9. 5

shRNA  siRNA template Termination sequence Promoter/enhancer • Hairpin siRNA • Pol III: string of 3-5 T’s • Pol III, (U6, H1) • Sense and antisense • Minimal SV40 poly A • Pol II, (CMV) signal strand of siRNA shRNA   Delivery    Plasmids    Viral vectors   Effect    Cell culture ‐ transient knockdown    In vivo ‐ long‐term (>1 week) effects  using viral vectors  Davidson BL, Paulson HL. Lancet Neurol. 2004 Mar;3(3):145-9. 6

MicroRNA (miRNA)   Endogenous RNAi pathway in animal cells   Endogenous ~21‐mer small RNA molecules from non‐coding RNA (introns,  independent miRNA genes)   Regulate gene transcription by binding to the 3’‐untranslated regions of  specific mRNAs   Key regulators of early development, cell proliferation, cell death,  apoptosis, cell differentiation, brain development   miRBASE ‐ http://microrna.sanger.ac.uk/     3000 miRNA sequences from various species    Sequences of many miRNAs are homologous between species     800 unique miRNAs in humans with 400‐500 conserved in mice    Regulate expression of at least 30% of protein‐coding genes  miRNA processing    Transcription of pri‐miRNA    Processing into pre‐miRNA in the  nucleus by Drosha    Export of pre‐miRNA to the  cytoplasm    Dicer complex processing    miRNA strand selection by RISC  http://www.ambion.com/techlib/resources/miRNA/mirna_pro.html 7

miRNA reduce steady state protein levels    Translational repression    Imperfect duplex      Reduces protein expression  without impacting on  corresponding mRNA  levels. (mechanism still  unclear)    mRNA degradation     Perfect duplex    Transcriptional regulation    guiding chromatin  methylation http://www.ambion.com/techlib/resources/miRNA/mirna_fun.html Using artificial miRNA as a tool for gene  function studies    Similar to applications for siRNA/shRNA    Invitrogen ‐ BLOCK‐iT™ system https://catalog.invitrogen.com /index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=12492 8

Potential Applications of RNA Interference   Testing hypotheses of gene function     Functional screening and target identification     Target validation for drug development     Potentially new therapeutic approaches to treating  diseases ‐ a new approach to antisense and new  possibilities for gene therapy  9

Gene therapy for Parkinson’s disease    Parkinson’s disease    Progressive neurodegenerative disorder affecting  ~1% of the population over the age of 65    Two types: sporadic and familial    Clinical symptoms: disorders in movement (resting  tremor, rigidity, akinesia, bradykinesia). Non‐motor  symptoms include depression and dementia.  Pathology   Selective degeneration of dopaminergic neurons within the substantia nigra  pars compacta (SNpc) that project axons to the striatum   Lewy bodies ‐abnormal protein aggregates in the cytoplasm of neurons   Dopamine depletion in the striatum causes disorders in movement   Focal pathology but has impact on overall basal ganglia circuitry  Adapted from Lindvall & Bjorklund. Nat. Med. 2000. 6, 1207-1208 1

What causes these dopamine cells to die?   Still largely unclear. Possible contributors:    Oxidative stress    Mitochondrial abnormalities    Excitotoxicity    Disturbances in calcium homeostasis    Toxins    Environmental e.g pesticides    Cellular e.g. dopamine, α‐synuclein  Animal models of PD    Models have predictive value with regard to dopamine deficiency     Rodent models     6‐hydroxydopamine (6‐OHDA) injected unilaterally into the  striatum or SNpc    MPTP (systemic injection) ‐ MPTP converted to toxic MPP+ which  is selectively taken up by dopamine neurons, inhibits  mitochondrial respiration     Rotenone (chronic infusion of low doses) ‐ mitochondrial complex  I inhibitor    transgenic mice (e.g α‐synuclein)  Non‐human primate     MPTP  2

Treatment strategies   Goals    Alleviate motor symptoms    Prevent ongoing cell death process in the SNpc   Current pharmacological treatments    Focus on augmenting striatal dopamine levels    L‐Dopa/carbidopa ‐ crosses the blood brain barrier and is  converted to dopamine by aromatic amino acid  decarboxylase (AADC)    Dopamine agonists and/or monoamine oxidase B inhibitors  which prevent dopamine breakdown, antioxidants,  glutamate antagonists may provide some benefits    Problems    Loss of efficacy over time, on‐off effects, dyskinesias,  hallucinations    Do not affect ongoing cell death process  3

Gene therapy strategies for PD    Three main strategies:    Biochemical augmentation ‐ alleviating symptoms    correct dopamine deficiency    Neuroprotection ‐ altering natural history of the  disease     growth factors (e.g. GDNF)    Resetting basal ganglia circuitry ‐ alleviating  symptoms/preventing further cell death?    silence overactive neuronal circuits by expressing glutamic  acid decarboxylase (GAD)  Biochemical augmentation   Express genes involved in dopamine biosynthesis    Tyrosine         Tyrosine hydroxylase (TH), tetrahydrobiopterin    L‐Dopa         AADC      dopamine   Vector systems used: HSV, Ad, AAV   Main disadvantage is inability to maintain dopamine concentrations  at appropriate therapeutic level   4

Neuroprotection    Introduce growth factor genes that promote cell survival and/ or regeneration of remaining neurons e.g. glial‐derived  neurotrophic factor (GDNF), brain‐derived neurotrophic factor  (BDNF), sonic hedgehog    Useful in early stages of the disease when there is still a  significant dopamine neuron population in the SNpc    GDNF gene therapy in particular, looks promising ‐  improvements in neurochemical assessments and motor  symptoms in rodent and primate models of PD using AAV and  lentiviral vector systems GDNF promotes survival and regeneration   GDNF promotes axon regeneration (sprouting) and correction of dopamine  deficiency in striatum   This effect only occurred when the vector was injected directly into the  striatum  Bjorklund et al., Brain Res., 886 (2000), 82-98 5

Methods used to assess whether gene therapy has  therapeutic effect and benefit    Neurochemistry ‐ measure dopamine levels (tissue  punches, microdialysis)    Assays for quantifying and/or visualising therapeutic  protein levels ‐ e.g immunohistochemistry, RT‐PCR,  Westerns, ELISA    Cell survival ‐ e.g TH as a marker of dopamine neurons    Non‐invasive imaging e.g. PET, MRI    Behavioural ‐ changes in motor function as a functional  endpoint  Functional assessment in rodents   Spontaneous motor activity   Drug‐induced motor activity ‐  rotational behaviour    Unilateral lesions produce an  imbalance in striatal dopamine  levels between the right and left  brain hemispheres and  upregulation of postsynaptic  dopamine receptors and/or signal  transduction sensitivity on the  lesioned side.  6

  Rate of rotation (e.g turns/min) is directly proportional to  severity of the lesion and dopamine loss.     Assess rotation behaviour before and after treatment.  Expectation is that if a therapy works, will see a reduction in  rotation rate.  Blue squares = AAVGDNF in striatum  Red squares = AAVGDNF in SN  Green = AAVGDNF in SN and striatum  Open squares = control  Bjorklund et al., Brain Res., 886 (2000), 82-98 Resetting brain circuitry   Dopamine deficiency in striatum has consequences on overall basal ganglia  circuitry   Overactivity in the subthalamic nucleus (STN)  7

Before deep brain stimulation  After deep‐brain stimulation  8

  Use a genetic approach to silence STN neurons by introducing  GAD (glutamic acid decarboxylase) ‐ enzyme involved in GABA  synthesis in these cells   Principle is similar to deep brain stimulation, an established  treatment for PD in humans   May also have a neuroprotective effect  GAD gene therapy in humans   World’s first gene therapy trial for PD approved by the U.S FDA  in 2003   Open label, safety and tolerability trial of AAV‐GAD injected  unilaterally into STN of 12 patients   First patient treated August 2003 with surgery on 12 patients  completed by July 2005   1 year follow‐up completed July 2006   Built‐in safety mechanism ‐ ablation of the STN is an  established treatment for PD.  9

Deep brain stimulation vs STN gene therapy  Study design  10

Functional assessment in humans ‐ GAD gene  therapy     Fluoro‐deoxyglucose PET imaging    MRI    Motor assessments ‐ Unified Parkinson’s disease rating scale  (UPDRS), Glosser QOL    Neuropsychological evaluation    Blood for haematology and chemistry, urinalysis, ECG    Blood for antibodies to AAV and GAD  GAD gene therapy led to improvement in  overall movement (UPDRS score)   “off “state “on” state 11

Improvement in movement on treated side  Functional PET imaging  12

Neural network activity discriminates PD and  controls Increased metabolism in SMA after GAD gene  therapy  13

Current status of PD gene therapy    Unravelling pathways involved in mediating death of  dopaminergic neurons will lead to identification of new  targets.    Areas to target: Ubiquitin‐proteosomal pathway (e.g parkin),  heat‐shock proteins   14

Gene therapy 2010

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Gene therapy 2010 Document Transcript