Possible biomedical applications of environmental biotechnology no kidding

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Possible biomedical applications of environmental
biotechnology -- no kidding! Aubrey D.N.J. de
Grey Department of Genetics, University of
Cambridge Email ag24_at_gen.cam.ac.uk Website
http//www.sens.org/
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Acknowledgements Preliminary data John Archer
(Cambridge), Ulf Brunk (Linköping) More recent
data John Schloendorn and colleagues (ASU),
Jacques Matthieu (Rice) Microbiology Bruce
Rittmann (Northwestern/ASU), Perry McCarty
(Stanford), Pedro Alvarez (Rice) Enzyme delivery
Ana Maria Cuervo (Albert Einstein), Roscoe
Brady (NINDS) Biomedical applications Ralph
Nixon (NYU), Jay Jerome (Vanderbilt), Janet
Sparrow (Columbia)
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• Structure of this talk
• Age-related intracellular aggregates and the
  evidence for their pathogenicity
• Bioremediation meets biomedicine
• Efficacy in principle
• Delivery
• Safety

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• Structure of this talk
• Age-related intracellular aggregates and the
  evidence for their pathogenicity
• Bioremediation meets biomedicine
• Efficacy in principle
• Delivery
• Safety

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Aggregates three major examples - A2E in macular
degeneration - Proteins in neurodegeneration -
Oxysterols in atherosclerosis
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Aggregates three major examples - A2E in macular
degeneration - Proteins in neurodegeneration -
Oxysterols in atherosclerosis
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Age-related accumulation of fluorescent compounds
in retinal pigment epithelium
neural retina
RPE
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Fundus autofluorescence (RPE lipofuscin)
increases with age
Exc. 550 nm
normal eyes 7 temporal to the fovea
Individually corrected for lens absorption
Delori et al., IOVS 421855. 2001
MOD FA
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RPE lipofuscin forms in photoreceptor outer
segments as a byproduct of the retinoid cycle
opsin
11-cis-retinal (vitamin A derivative)
visual cycle
lipofuscin fluorophores
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Amphiphilic compound
2 hydrophobic side-arms
cationic polar head
Cl -
iso-A2E
A2E
detergent-like activity
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Detecting A2E-epoxides by mass spectroscopy
A2E bis-epoxide
A2E
624
640
A2E nona-epoxide
608
656
672
FAB-MS
592
688
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704
720
736
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oxidation of DNA bases
modifications of protein
changes in gene expression
apoptosis
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Aggregates three major examples - A2E in macular
degeneration - Proteins in neurodegeneration -
Oxysterols in atherosclerosis
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Autophagy in Alzheimers Disease
Dystrophic Neurites
IEM
Calnexin
Cat D
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Autophagy in Dystrophic Neurites
Autophagosomes pH 7.4
Autophagolysosomes LEP100 pH lt 6.7
Dystrophic Neurite
Lysosomes
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• Can lysosomal accumulation of anything possibly
  be beneficial?
• Model 1 aggregation of misfolded proteins is bad
  (prevents their digestion by cytosolic proteases
  or chaperone-mediated autophagy)
• Inference lysosomal aggregates result from
  macro- or microautophagy of cytosolic aggregates
  followed by failure of their lysosomal
  proteolysis
• Thus intralysosomal accumulation is bad

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• Can lysosomal accumulation of anything possibly
  be beneficial?
• Model 2 aggregation of misfolded proteins is
  good, as a staging-post when their proteolysis is
  failing aggregates are autophagocytosed when
  possible
• Inference lysosomal aggregates result from CM-,
  macro- or microautophagy of cytosolic proteins
  followed by failure of their lysosomal
  proteolysis
• Thus intralysosomal accumulation is bad

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• Can lysosomal accumulation of anything possibly
  be beneficial?
• Model 3 aggregation of misfolded proteins is
  good, because aggregates nucleate more misfolded
  proteins and thus clear them faster
• Inference lysosomal aggregates result from CM-,
  macro- or microautophagy of other molecules,
  which cause failure of lysosomal proteolysis
• Thus intralysosomal accumulation is bad

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• Can lysosomal accumulation of anything possibly
  be beneficial?
• Nothing that gets into lysosomes gets out again
• Lysosomal aggregates are biochemically inert

Thus intralysosomal accumulation is bad
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Aggregates three major examples - A2E in macular
degeneration - Proteins in neurodegeneration -
Oxysterols in atherosclerosis
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Atherosclerosis Progression
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Atherosclerotic Lesion
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Foam Cell Formed by Receptor-Mediated
internalization of Low Density Lipoproteins (LDL).
LDL
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LDL
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LIPOPROTEIN
Chol-E
Endocytosis
LYSOSOME/LATE ENDOSOME
Chol-E
LAL
Chol-E
Chol FA
FA
Plasma Membrane
ACAT
Chol-E
Chol
Chol
NCEH
Cholesterol Efflux Promoter
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Atherosclerotic Lesion Composition
Monocytes
Endothelium
Lipoprotein
Macrophage Foam Cell
Smooth Muscle
Primary lipid in lesion is Cholesterol and
Cholesteryl Esters
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Endothelial Cells
Lipid-engorged Lysosome
Foam Cell
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OxLDL-treated Human Macrophages accumulate FC and
CE in lysosomes
Acid Phosphatase Stained
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Known effects of Oxidation of LDL on Lysosomal
Function

• Heavily oxidized LDL will directly inhibit
  cathepsins and lipases.
• However, does not produce cellular cholesterol
  accumulation because cholesterol converted to
  oxysterol
• Heavily oxidized LDL is cytotoxic
• Modestly oxidized LDL promotes lysosomal
  cholesterol accumulation.

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Possible Lipid-Induced alterations in Lysosomes

• Direct inhibition of enzymes
• Inhibition of delivery of enzyme to lysosome
• No inhibition of delivery of endocytosed
  lipoprotein
• Some evidence of disruption of trafficking
  between lysosomes and TGN
• Alteration in the lysosomal environment

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• Structure of this talk
• Age-related intracellular aggregates and the
  evidence for their pathogenicity
• Bioremediation meets biomedicine
• Efficacy in principle
• Delivery
• Safety

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• Bioremediation the concept
• - Microbes, like all life, need an ecological
  niche
• - Some get it by brawn (growing very fast)
• - Some by brain (living off material than others
  can't)
• Any abundant, energy-rich organic material that
  is hard to degrade thus provides selective
  pressure to evolve the machinery to degrade it
• - That selective pressure works. Even TNT, PCBs

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Example DGGE Results from Perchlorate-Reducing,
Membrane Biofilm Reactors
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Xenocatabolism the concept Graveyards -
are abundant in human remains - accumulate
bones (which are not energy-rich) - do not
accumulate oxysterols, A2E etc... - so,
should harbour microbes that degrade them -
whose catabolic enzymes could be therapeutic
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Environmental decontamination in vivo
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• Steps to biomedical application
• Isolate competent strains select by starvation
• Identify the enzymes (mutagenesis, chemistry,
  genomics)
• Make lysosome-targeted transgenes, assay cell
  toxicity
• Assay competence in vitro (more
  mutagenesis/selection)
• Construct transgenic mice, assay toxicity in vivo
• Assay competence in disease mouse models
• Test in humans as for lysosomal storage diseases

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• Structure of this talk
• Age-related intracellular aggregates and the
  evidence for their pathogenicity
• Bioremediation meets biomedicine
• Efficacy in principle
• Delivery
• Safety

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This might just work preliminary data
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This might just work preliminary data
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7-ketocholesterol degradation - a promising start
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Identifying the genes - first steps
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• Other major efficacy issues
• - How many enzymes would we need?
• Maybe not many LSDs (single-gene disorders)
  imply big synergy between the various enzymes
• - How could we make them work in mammals?
• LacZ does but also, in vitro evolution fungi
• What about low-abundance lysosomal toxins?
• Abundance is presumably not that low

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• Structure of this talk
• Age-related intracellular aggregates and the
  evidence for their pathogenicity
• Bioremediation meets biomedicine
• Efficacy in principle
• Delivery
• Safety

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• How can we get these enzymes to lysosomes of
  affected cells?
• Gene therapy
• Stem cell therapy
• Enzyme therapy

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Main Trafficking Pathways
Vesicular Traffic
Chaperone-mediated Autophagy
CVT Macroautophagy
Modified from Alberts 2002
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• How can we get these enzymes to lysosomes of
  affected cells?
• Gene therapy
• Stem cell therapy
• Enzyme therapy

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Amino Acid Chain
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Amino Acid Chain
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• Structure of this talk
• Age-related intracellular aggregates and the
  evidence for their pathogenicity
• Bioremediation meets biomedicine
• Efficacy in principle
• Delivery
• Safety

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• Safety the main issues
• Immunogenicity?
• Tolerisation brief expression low expression
• Extralysosomal toxicity?
• Specificity coding as proenzymes try again!

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• Conclusions
• Disparate areas of biology can converge (if
  anyone has time to spot the link!)
• We need to try radical approaches
• This might just work..