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84 Cards in this Set
- Front
- Back
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Gene therapy
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delivery of DNA to correct a genetic defect (replacement therapy)
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Vectors
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modified (removed pathogenicity) viral vectors deliver genes to nuclei
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Tropism
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specific cell target of a virus- vector utilization relies on tropism matching
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Requirements of vector delivery
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deliver cDNA to target tissues along with important regulatory information (promoter driven by transcription factor expressed in the target cell population)
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Advantages of gene therapy
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direct replacement of defective genes
minimal side effects if successful theoretically applicable to all genetic diseases |
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Disadvantages of gene therapy
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Viral delivery: requires optimized tropism, limited carrying capacity
Immune response to the viral capsid and transgene product Insertional mutagenesis |
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Tropism of rhinovirus
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respiratory system
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Dystrophin can't be delivered in AAV because
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Dystrophin (14kb) exceeds the carrying capacity of adeno-associated viral vector (3kb)
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Becker dystrophin mutation
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truncated protein (50%) is still somewhat functional
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Western blot analysis of the truncated dystrophin reveals
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-retains n-terminal, spectrin repeats and c-terminal
-Faint bands at 200kb recognized by Ab for 400 kb segment -Ab for spectrin repeats do not bind |
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Conclusion of western blot for dystrophin gene
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deletion covering half the gene but retaining n and c terminals is critical for function, some sections of the gene are redundant
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Mini-dystrophin
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22kDa; expression of microdystrophin transforms phenotype to less severe MD
Has alternative splicing |
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Fixing the gene means
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manipulate gene expression by alternative splicing and exon skipping
occurs at the pre-mRNA level |
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Why is DNA used instead of RNA when fixing a gene through alternative splicing and exon skipping?
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DNA is used instead of RNA because RNA is highly degradable and sequence preservation requires a more stable nucleic acid backbone
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Exon skipping is induced by...
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Anti-sense oligos are hybridized with a particular region of the exon or the border between an exon and intron resulting in the translation of a complete protein without the mutated nonsense mutation
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Agents of exon skipping
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2' OMe-AON or PMO (morpholino)
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Drisapersen (created by GSK)
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six minute walk test did not demonstrate a significant clinical change and toxicity lead to kidney problems
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Eteplirsen (crated by Sarepta)
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did not demonstrate toxicity effects but the sample size was too small to gain FDA approval
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Types of mutations
-silent -missense -nonsense -frameshift |
-silent: codons do not change
-missense: codon change -nonsense: code for a premature stop codon -frameshift: insertion or deletion of a base causing all following codons to shift |
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Why is inducing read through possible without disrupting normal translation?
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Premature stops utilize different machinery
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Gentamicin
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antibiotic which induces read through but high doses can cause hearing loss
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Ataluren (PTC 124)
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induces read through effectively but ambiguous results through all three trail phases (saw trends but did not reach statistical significance)
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Difference between immunohistochemistry and western blots
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immunohistochemistry: does not show size but shows expression levels
Western blots: show size but not expression levels and not localization |
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Clinical trials and phases
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Demonstrate efficacy and safety of a drug while measuring clinical change
Phase I: Safety Phase 2: Dosage studies on target and biological efficacy Phase 3: efficacy and safety in a large population |
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Endogenous mechanical pathway is mediated...
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by myostatin (GDF-8: negative regulator of muscle mass)
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Growth factor 8 (GDF-8)
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binds to activin receptor type II to inhibit the growth of muscle tissue
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Inhibition of GDF-8
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Binding to ACE-031 (neutralization)
Example of fc-fusion using fc-act RIIb |
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Why is fc fusion necessary for inhibiting GDF-8
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Necessary for stability and manufacturing purposes but does not have specific functionality for inhibiting this pathway
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Why can't we use classical antagonists to inhibit GDF-8 myostatin pathway
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There aren't any antagonists for the activin receptor type II and the signalling pathway is too common to allow for specific inhibition without inducing adverse side effects
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Bulking up muscle to support muscular dystrophies is an example of...
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adjunct therapy (does not treat the underlying cause, not a disease modifying therapy)
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Stem cell sources
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Progenitor cells (differentiate in a controlled manner) from resident stem cells and mesoangioblasts (pericytes and satellite cells)
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Induced pluripotent stem cells
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autologous and remove the major obstacle of immune responses but require the defect to be corrected before integration by viral mediation or gene editing
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Major concerns for stem cell therapy
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-immune response caused by HLA haplotypes
-niche are degraded during muscle degeneration -difficulties during integration |
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Epilepsy
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a variety of neurological problems under a single rubric
a tendency toward recurrent seizures unprovoked by systemic or neurological insults |
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Seizure
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clinical manifestation of abnormal and excessive excitation of a population of cortical neurons
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Epilepsy as chronic condition
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individuals can have seizures without being epileptic because brains are hardwired on the edge disaster therefore epilepsy is characterized by recurrence
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Epileptogenesis
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a sequence of events that converts a neuronal network into a hyper-excitable, seizure prone network
-Genetic basis contributes but the majority of cases are spontaneous or the result of neuronal insults |
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EEG
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non-invasive recording of the electric activity of the brain
amplitude records are dampened by insulating layers Small signals: desynchronized activity |
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Seizures on the EEG
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characterized by increased amplitude and highly synchronized activity of the entire cortex
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Absence seizure
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subtle physical manifestations and disconnected from the stimulus
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Generalized seizure
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takes over the whole brain
can be primary generalized or secondarily generalized, convulsive or non-convulsive |
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Distinction between primary generalized and secondarily generalized
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requires knowledge of patterns of onset and may be predicted on clinical or EEG data
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Convulsive seizures
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generalized tonic-clonic, myoclonic, tonic and atonic
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Partial seizures
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identifiable location of seizure initiation which can stay localized or generalize
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simple seizures
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localized, no loss of consciousness
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complex seizures
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unilateral but alters consciousness, awareness and responsiveness
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Prevalence and incidence
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strongly dependent on age with higher rates among children and elderly
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Relationship of etiology to age
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First two decades: genetics and early life CNS insults
Elderly: vascular, neoplasia, neurodegenerative, toxic, metabolic causes or withdrawal |
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Drawbacks for EEG and seizures
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a large percentage of seizures are generated from parts of the brain that are hidden from EEG electrodes specifically the medial temporal lobe (especially the hippocampal region)
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MEG
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measures minute magnetic fields in the brain from a population of neurons
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EEG vs. MEG
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MEG is deeper, more sensitive and faster than EEG but the machines are more expensive
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Seizures are symptoms of underlying pathological...
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increased excitation, decreased inhibition, neurons with changed physiology or structure, too many neuron connection
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Pyramidal cells
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excitatory neurons are the excitatory neurons of the cerebral cortex, highly interconnected allowing for cascading signals to propagate and amplify.
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Seizures and pyramidal cells
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Seizures can be thought of as unusually synchronized firing of pyramidal cells
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genetics of epilepsy
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many mutations that affect GABA and a number of not well understood genes relating to ion channnels
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method of epilepsy treatment
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vagus nerve simulation works as a pacemaker through unknown pathology
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the hope for epilepsy treatment
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closed loop control through trans-cranial electrical stimulation
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Allostasis
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adaptive response to stressors to maintain homeostasis
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Adrenals
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important for regulating repair and general function
endocrine signaling through the bloodstream (stress hormones) |
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Corticosterone and noradrenaline
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steroid and catecholamine/NT
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Effect of adrenals
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increased respiration, pupil dilation, increased heart rate, bronchial dilation, mobilization of sugar from fat, vasoconstriction
Decreased irrelevant function (digestion, saliva, immune) |
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Stress response goals
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short term goals: survive, mobilize resources, enhance senses, stop building projeccts
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Classical definition of stress
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organization and mobilization of biological processes to promote survival (HPA-axis response)
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Neuroanatomy of stress
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Evaluation: mPFC
Emote: AMG Associate: HC Hypothalamus (AMG) acts as the big red button |
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HPA axis
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HC releases CRH -->
Anterior pituitary releases ACTH --> Adrenals are impacted by endocrine signalling |
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corticotrophin release hormone
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hormones that act on the cortex
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adrenocortiotrophic hormone
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adrenals that act on the cortex
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Elevation of cortisol
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affects consolidation and learning
promotes neuronal growth control of gene expression (signaling and learning) |
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MR and GC
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mineralcorticoid- binds with high affinity
glucocorticoid- binds with low affinity |
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Effect of corticosterone
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acts on MR and GC
enhanced signaling (blocks GABA, increase ca2+ and glut release) |
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Excess stress
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blocks NMDA, prevents BDNF, associative plasticity and suppresses ca2+
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Extreme/chronic stress
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atrophy of spines, shrinkage of prefrontal cortex and HC
(long term system that are metabolically demanding) |
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Human stress
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Natural stress events are normally acute (no way to handle chronic stress) and driven with associative centers (psychological not physiological)
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Reversing stress effects
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most of the effects of stress are reversible even in adult life (recovery of HC and PFC) but AMG effects are typically irreversible.
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Protracted development of stress
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big AMG and small HC (fear conditioning and extinction)
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Kasper-hauser syndrome
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psychological dwarfism (mental retardation and cognitive delays)
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Adverse childhood experiences
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cause a whole host of psychological and physiological disorders
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Defenses against chronic stress
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early stress hormones- matures nervous system quicker, changes in gene expression leading to a more adult stress response
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Animal models of stress
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induce stressful maternal behaviors- study the effect of stress in
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Observation of stress in animal models
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increased anxiety, depression, impaired learning, enhanced addition, increased seizure, aggression
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Effects of ELS
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early stress hormones: BDNF, corticosterone/cortisol
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Fear condition
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contextual- hippocampus
cued: amygdala fear extinction learning: prefrontal cortex |
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BDNF
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signaling molecule that leads to elaborate dendritic arbors
- cell proliferation, migration, curvival and plasticity - dynamic expression over development - gene expression and mutation mimic ELS - chronic stress decrease BDNF |
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Mood stabilizers
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that augment BDNF have an improved outcome
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