Presentations day 1

what are HERVs?

• human endogenous retroviruses → make up 8% of human genome

• mutations/recomb b/w LTRs → loss of replicability → 10% due to buildup of mutations, 90% LTR leaving (middle lost)

why aren’t HERVs constitutively expressed?

silencing via epigenetic modifications → to prevent expression of viral promoters

• methylation of CpG (cysteine, guaning) islands → histone recruitment

  • works with histone deacetylase to clump DNA to prevent expression

  • when many methyl groups added to CpG repeats, steric hinderance blocks gene replication

how do HERV’s act as a tumor marker and what does that have to do with ‘viral mimicry’?

• certain cancers (eg. seminoma) interrupt CpG methylation processes → relief of HERV suppression in cancer cells = increased HERV expression → decreased methylation = increased HERV exp

• HERV expression leads to antiviral response

  • dsRNA → IFN → ISGs

  • shown by RNAseq

what are the effects of increased ISG expression with HERVs?

• upregulation of MHC I /TAA → good

• release of chemokines → good

• upregulation of PD-L1/CTLA-4 → not good, decreases T cell activity

what are DNMTi drugs?

DNA methyltransferase inhibitors

• cytosine analogues

• covalently ‘trap’ DNMT at CpG dinucleotides at the replication fork → trap enzyme, blocking further activity → effective at low doses

• enzyme normally allows switch from proton to electron → nucleophilic attack, methyl attaches

• drug has N where C would usually be that would be methylated → when enzyme attacks N gets covalently trapped

describe ICIs as cancer therapies

• ‘immune checkpoint inhibitors’ relieve inhibition of T cells by tumor cells

• target inhibitory immune checkpoint receptors on tumor cells → PD-L1, CTLA-4)

• can also target ligands on T cells → PD-1, CD80/86

• small antibody mol → bind to receptors, block signalling

how could HERVs and the ICIs be combined?

• use DNMTis alongside ICIs → inhibiting methylation would theorhetically increase HERV expression → increase HERV get increase PD-L1 → more targets for ICI → ICI more efficient

• saw decreased tumor burden when combined, but doesn’t directly show due to HERVs specifically

what are the promising features and challenges with exploiting HERVs to improve immunotherapy efficacy

promising features:

• reversal of T cell exclusionary cancerous phenotypes (‘cold’ tumors → ‘hot tumors’)

• DNMTi ‘priming’ restores responsiveness to aPD-1 therapy → allow histones chromatin to break apart & activate path?

• many clinical trials underway

challenges:

• variable results between cancer types and patients → blocked IFN prod = blocked DNMTi action. immunosuppression = impaired action

• neutralization of ‘good’ viral elements → cleared by IS

• chronic antiviral signalling = immunosuppressive

what is hyperthermia cancer therapy?

• hyperthermia = increasing temp → 39-44C

• increases tumour susceptibility → cooks the cell

• more targeted chemotherapy → thermoresponsive liposomes

• specificity can be improved

what virus is used as part of the hyperthermia presentation?

• macrobrachium rosenbergii nodavirus → using VLP

• freshwater prawn virus

• capsid protein can form VLPs

• capsid stability forms in association with RNA → like HBV, need stable assoc.

• many chemo drugs bind RNA → can bind chemo drug to RNA then form capsid around it

how did they target cancer cells in the heat-seeking VLP treatment

• folate receptor is overexpressed in ~40% of cancers, as folate is required for dNTP (purine) synthesis

• add folic acid to the surface of VLP to act as receptor-binding protein

describe how the heat-seaking VLP treatment works

add folic acid to surface of MrNVLP → package Dox, a nucleic acid chelating agent, inside by binding incorporated E. coli RNA → destabilize Dox from virus only when heat is added

• showed temp sensitive

• showed VLPs are specific for folate receptors → VLPs more specific for cells expressing high levels of folate receptors

what are the promising features of MrNVLP-Dox?

• no seroprevalence → freshwater prawn virus

• no/limited Ab neutralization → folate (aka vitamin B12) covers whole VLP so can’t access capsid

• many chemotherapies can bind RNA stably

• can easily add diff receptors for diff cancers

• lowers risk of off-target effects

• potential Ag presentation on tumors of VLP

• immune activation from RNA → E. coli RNA interact w/ PRRs

what are the challenges associated with MrNVLP-Dox

• no in vivo experiments shown

• could make it easier for Dox to enter healthy cells → female reproductive tissues increase expression during shedding cycle, macrophages express it too

• Dox can slowly release from VLP at 37C → likely require intratumoral administration

what does Arc do?

downregulates synaptic signalling

• neuronal protein expressed in response to synaptic activity

• enriched at excitatory synapses

• Arc capsids may be required to eliminate synaptic material

• required for learning and memory processes

• Arc capsids endocytosed, Arc mRNA released, binds AMPARs, which get endocytosed and dampens activity of neuron

describe Arc’s N lobe

• Arc’s N-terminal lobe has a:

  • hydrophobic pocket

  • Beta1 (B1) strand

• B1 mediates binding to synaptic proteins

  • Stargazin → AMPA receptor trafficking, synapitc regulation

where does Arc originate from?

• Ty3/gypsy retrotransposon

  • encodes protein similar to gag’s capsid (CA) protein → related to retroviruses

• domestication of retrotransposon-derived gene → lost ability to form infectious particle (lost zinc knuckles), was repurposed

how does Arc compare to HIV protein?

Arc superimposes on Gag’s CA domain → HIV C-lobe

• function: self-assembly into capsid, RNA packaging, intracellular delivery

• but sequences aren’t super similar → but residues forming hydrophobic pockekt are conserved

why is there a need for studying Arc?

• implicated in cognitive diseases → schizophrenia, autism, alzheimers

  • understanding its action

  • binding properties

• therapeutic potential to modulate: synaptic plasticity, memory pathways

• novel mechanism of intracellular communication → sharing of genetic material

generally describe how Arc is hypothesized to function

1. Arc self-assembles into viral-like capsids in the donor neuron

2. Arc mRNA packed into capsid

3. which are released from donor cells in extracellular vesicles dubbed ACBARs

4. mRNA released into recipient neuron to be translated and continue down path to inhibit neuronal activity

what/how did they show Arc’s functions?

• showed Arc self-assembles into virus-like capsids using its full gag-related genome → CA domain not sufficient for assembly, self-assemble spontaneously

• Arc capsid can transfer Arc mRNA between neurons → incubated hippocampal neurons from Arc KO mice with purified rat Arc → measured Arc mRNA levels in Arc KO mice using Arc FISH, then incubated with prArc

what makes Arc work/knowledge promising?

• a tool to map memory engrams (in use)

  • tightly linked to neuronal activity during learning

  • marks neurons involved in specific experiences

• a neuron-specific RNA delivery system → endogenous

• targeting Arc to treat cognitive disorders

  • Arc dysregulation → synaptic dysfunction?

  • pharmacological modulation

what makes Arc work challenging?

• risk of triggering immune response in brain

  • oligomeric particles, highly repetitive & symmetric

  • package RNA → TLR → inflammation in brain → neural degeneration"?

• delivery beyond neural circuits

  • cargo specificity

  • targeting

  • high scale production?

what is the current problem with how we trace neural circuits? what do we need?

• brain has 100 billion neurons and 1 quadrillion synapses → very complex system

need a tool that can:

1. trace connections across synapses

2. identify directionality

3. label specific neurons

4. link structure to function

compare non-viral vs viral tracers

non-viral: chemical, eg. silver stain → stains well but can’t distingush individual synapses

• rely on injection

• label non-specifically

• label “axons of passage” → false positives, intermingled rather than connections

• no directional control

viral tracers:

• cell type specificity

• can separate from intermingled neurons

• trans-synaptic labelling

• directional control

why is rabies a good candidate for neural tracing?

enveloped & bullet shaped

• easy to genetically engineer envelope proteins

• bullet structure → efficient travel across long distances

retrograde & neurotropic

• moves backwards from synapse towards cell body → exclusive directional control

• naturally infects neurons

trans-synaptic spread

• specific spread across junctions, rather than non-specific diffusion

immune evasion

• can persist in body without being cleared

describe the first gen rabies neural tracers

• had deletions in the envelope glycoprotein → G protein is key to entering cell, without it won’t spread well

• problems:

  • cytotoxic

  • kill neurons within 2 weeks

  • impacts endogenous gene expression

  • short-term, static visualization

describe the second gen rabies neural tracers

• deletion of the glycoprotein and large polymerase gene → don’t spread, don’t replicate

• features:

  • 90% neuron survival

  • preserved physiological integrity

  • long term imagine

  • regulatable viral replication

describe the machanism behind the rabies neural tracing

can precisely target cells using the EnvA and TVA system:

• pseudotype rabies with EnvA

• mammalian neurons lack TVA recepto

• starter cells genetically engineered to express TVA + G protein via AAV vector → prgeny rabies goated with G protein and can move to neighbouring cells

• infection becomes cell-type specific

what results were shown for the rabies neural tracing?

• first gen is fast but toxic, no source cells left

• very few labelled, initially, after 5 weks label appears in thalamus, secondary motor cortex, somatosensory, doxycycline suppresses G and L → turns off viral replication and spread

• showed that corticostratial neurons are integrating inputs from opposite cortex an thalamus → evidence of cross-tall

what are the applications of the rabies neural circuit tracing

• identified the presynaptic inputs to dopamine neurons in the ventral tegmental area in mice → region involved in reward and motivation

• found that VTA dopamine neurons receive input from over 20 brain regions

• map spacial organizations

what are the challenges with rabies neural circuit tracing?

• neurotoxicity → limits range of species that can be tested

• possible synapse bias → may preferentially infect inhibitory vs excitatory neurons, study using first gen showed no bias but no studies done on second gen

• possible transport limitations → distant neurons may not be getting labeled, slow or incomplete viral transport across long distances

what are the promising features of the rabies neural circuit tracing

• 3rd gen tracer → some studies being done with dL version, even better labelling

• gene manipulation → design inhibitors/agonists that may manipulate the behaviour of infected neurons

• neuron studies → could monitor change in neural connections over time, what areas degrade faster, brain injury studies, smell/scent studies…