Lecture #20

Treatment of eye disease4/11/13

Next week

• Create your first pageIntroduction to topic and why it is important

Doesn’t have to be final versionPicture to illustrate your topicList of 3-5 references that will be key to your project

Finding references

• PubmedSelect ReviewsFilter for free availabilitySome of the journals will be available through our library

Request any you need from ILL - NOW

Naming pages uniquely

• Name your pages with a unique name (perhaps including your initials)

• References subpageIf everyone makes a page called “References” they will write over each other

Wiki - Table of contents

• If at least four headers on a page table of contents (TOC) appears in front of the first header (or after introductory sections). Putting __TOC__ anywhere forces the TOC to appear (that is two underscores _ _ before and after)Putting __NOTOC__ anywhere forces the TOC to disappear.

Sources

• Use primary referencesTry not to use other web pages

Telephone gameIll try to help find a few papers for each topic

For Thursday next week

• Make your introductory pageHow organize home page

• Identify 3-5 referencesPut on intro page or stubs for other pages

Gene therapy

The future promise of curing all disease

Gene therapy

• Many diseases of the eye are the result of single gene mutationsOver 200 genes now knownRhodopsin, phototransduction pathway, visual cycle

• If you could insert a gene to repair damage - the disease would be cured

Ideal scenario for gene therapy

• Know the mutated gene to replace• Have good copy of gene• Understand biology

Know tissue and how gene is acting• Know that adding gene back will solve

problem• The eye is a contained organ so therapy does

not impact other organs

Ocular gene therapy

• Three kinds of therapiesIntroduce a gene to make a protein which alleviates some of symptomsIntroduce a replacement gene to fix mutationIntroduce a gene to knock out faulty gene (RNAi)

What is needed to make this happen?

• Gene expressed to sufficient extentMake enough “stuff” to provide relief

• Gene expressed for long timeRetinal cells don’t divide so doesn’t need to integrate with DNABut does need strong promoter so expressed in tissue of interest

• No cause of inflammatory or immunogenic response

Vectors for gene delivery - Viruses

• ProsExisting method for getting DNA into cells

May be engineered to target particular cellsCan be modified so don’t replicate and destroy cell

• ConsVirus can only handle a gene up to certain sizeMay trigger immune response making person sickPerson may develop immunity so virus gets destroyed

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Alqawlaq et al 2012

Getting gene to tissue of interest

• In vivoAdd directly to tissue in the body

• Ex vivoRemove cells of interestCulture cellsAdd vector containing gene of interestIf gene integrates, add back to body

Advantages of viruses

• Common in humans43 different types

• Rapidly infect many kinds of human cells with high gene transfer rate

• Low pathogenicity• Can hold up to 7.5 kb of DNA• Viral DNA is stable with no rearranging• Viral DNA is easy to manipulate

Gene therapy with adenovirus

Nature Genetics 28: 92 (2001)

Visual cycle (lecture 16)

RPE 65 is key isomerase in RPE to convert all trans retinal ester to 11-cis retinolMutations cause Leber congenital amaurosis

Dog model : Swedish Briard• Have mutation in RPE65

4 bp deletionCongenital stationary night blindness

Congenital - from birthStationary - stableNight blindness - affects rods

Can also have some degeneration with time

First show can treat RPE cells with adeno-associated virus containing RPE65 to rescue mutant RPE65-/-

WT RPE cels RPE65-/- AAV treated

Nuclei are orange from propidium iodide stainingRPE65 antibody glows green

WT retina RPE65-/-

In vivo treatment - Divide eye into retinal quadrants

Inject AAV-RPE65 into TS

TSTemporal-superior

TI Temporal inferior

NINasal inferior

NSNasal superior

PCR of DNA from wild type and mutant RPE65

Wild type

Mutant w/ 4 bp deletion

109 bp

105 bp

Use PCR to screen for expressed gene after innoculate with AAV - RPE65

Persistent expression of new RPE65 form (99 days after inject)

RPE65

Individual dogs: WT, hetero- and homozygous mutant

Cultured RPE cells WT, mutant pre- and post-treatment

Use PCR to screen for expressed gene after innoculate with AAV - RPE65

RPE65

R=retinaP=RPE

In vivo treatment. See injected functional RPE65 in TS region only Not in other 3 eye quadrants

Use PCR to screen for expressed gene after innoculate with AAV - RPE65

Get expressed cDNA only in RPE of quadrant where injection occurred.

RPE65

RT PCR / cDNA

genomic

Electroretinograms show improvement resulting from AAV-RPE65

Downward a wave from photoreceptorsUpward b wave from bipolars, oscillations from amarcrine

Injections must be subretinal

Intravitreal injections don’t work

Video by Acland et al showing dog behavior

Subretinal injection of AAV-RPE65 gene

Retinal photoreceptor distributions before treatment

PR thickness

Light sensitive area increases as does light sensitivity by:

P1 10xP2 100xP3 1000x

Time after injection1 month2 months3 months

Enhanced light sensitivity to that expected based on number of photoreceptors they have

So 11-cis retinal supply is much better!

Retinal gene therapy

• Is well on its way!

Transgenic animals to enhance color processing

Jeremy Nathans• Professor, Johns Hopkins and

HHMIBS Chemistry and Biology MITPhD Biochemistry and MD, Stanford

• Sequenced the bovine rhodopsin gene

• Sequence the human rod and cone opsin genes

Gerald Jacobs• Professor, UCSB

BA U VermontPhD Indiana UAsst Prof UT AustinUCSB starting in 1969

• Electrophysiology, psychophysics• Human and primate red/green

vision

Normal mouse visual pigments

UV cone - 360 nm

Green cone - 508 nm

Engineered a new mouse where green gene replaced by red

UV cone - 365 nmRed cone - 565 nm

If cross mice will get homozygous..

…and heterozygous mice

Just like primates

• Different alleles on X chromosome• Females can be heterozygous• X inactivation will result in some cones

expressing green gene and some expressing the red gene

• Does this enable enhanced color vision?????

Test spectral sensitivity of different mice

• Use electroretinogram to measure sensitivity of entire retina

• For heterozygous mice can estimate the fraction of cones which have L pigment

512 nmn=12

556 nm n=17

n=87

Heterozygous mice

• Get range of L:M cone ratios in heterozygotes

• Differences in X inactivation and / or expression?

Behavioral testing

Operant training - mouse gets drop of soy milk as reward for choosing the light that differs from the other two

Do L cones contribute to light detection?

• Compare thresholds at which can see difference from achromatic backgrounds

Add different amounts of either 500 nm or 600 nm light What is threshold needed to distinguish it from white?

Compare threshold at 500nm and 600nm

Mice with M+L cones more sensitive to 600 nm light than those with only M

How much more red light vs green light is needed to get behavioral response Threshold difference report as Log 500 nm/600nm

If log x = 1 then x = 101

Mice with M+L cones more sensitive to 600 nm light than those with only M

So if only M cones, response requires 101.3=20x more 600 nm than 500 nm light

If M+L cones, response requires only 10.85= 7x more 600 nm light than 500 nm light

So L cone makes more sensitive to 600 nm light

Conclusion

• New L cones do contribute to the detection of light.

• Do they contribute to color vision??

Test color discrimination

Have two targets with 600 nm light.Third is illuminated with various wavelengths. If mouse can’t tell those from 600 nm, then mouse will guess randomly which target is correct.

% correct choices when compare light at test wavelength with 600 nm

M only

M:L 78:22

M:L 65:35

Need significant # of L cones to discriminate colors

M only

M:L 78:22

M:L 65:35

M:L 44:56

M:L 46:54

M:L 53:47

Color mixing experiments

• Vary amount of 530 and 620 nm light shine on same target

•When can mouse tell this mixture apart from 600 nm?

530 nm 620 nm

Color matching results

Mouse can tell mixture apart until get close to pure 600 nmHuman observer compared to prediction also shown.

What do you think?

• If give mice a third pigment, do they become trichromats?

• What more proof could be gathered?

Alternative interpretation

Mouse view

• Might not see color - might see varying brightness

Homozygous mouse

Heterozygous mouse

500 nm 500+600 nm

Neitz’s repeat experiment in monkey

Dichromatic male squirrel monkeys

Start with males who are dichromates Blue and green cones

Some females are already trichromats so don’t use them

Trichromats

Gene therapy with LWS gene

• Treat adultsNo change in wiring - would it be utilized?If works, set stage for treating adults with occular disease using gene therapy

• Subretinal injection of recombinant adeno-associated virus containing human red opsin

Response to red light using ERG

Retinal map 40 wks after two injections

Inset shows 16 wks after so takes a long while for Lops to be expressed

Response to red light using ERG

Retinal map 40 wks after two injections

Coinjected GFP viruses

Estimate 15-36% of cones are LWS

Cambridge color vision test

Dichromats confuse certain colors with grey

Hues that stimulate both cones equally

Cyan = Blue-green stimulates both cones equally.Gray also stimulates both cones equallySo hues near the pigment crossing points are hard to distinguish from gray

Dalton - squirrel monkey

Ask Dalton to locate colored square

Need to distinguish it from gray

Get reward if correct

Cambridge vision test modified for animals

Can increase the saturation of color to see at what threshold it is distinct from gray

Possible outcomes of Lops addition

Could shift sensitivity to longer wavelength

Could remove uncertainties so can discriminate all colors

Squirrel monkey before and after treatment with virus containing

Human LWS gene