DODGE (2008) Delivery of AAV-IGF-1 to the CNS Extends

8/8/2019 DODGE (2008) Delivery of AAV-IGF-1 to the CNS Extends

1/9

The American Society o Gene Therapyoriginal article

1056 www.moleculartherapy.org vol.16no.6,10561064june2008

Delivery o AAV-IGF-1 to the CNS Extends Survivalin ALS Mice Through Modifcation o Aberrant

Glial Cell ActivityJames C Dodge1, Amanda M Haidet2,3, Wendy Yang1, Marco A Passini1, Mark Hester2, Jennier Clarke1,Eric M Roskelley1, Christopher M Treleaven1, Liza Rizo2, Heather Martin2, Soo H Kim2,3, Rita Kaspar2,3,Tatyana V Taksir1, Denise A Griths1, Seng H Cheng1, Lamya S Shihabuddin1 and Brian K Kaspar2,3

1Genzyme Corporation, Framingham, Massachusetts, USA; 2Center for Gene Therapy, The Research Institute at Nationwide Childrens Hospital,Columbus, Ohio, USA; 3Integrated Biomedical Science and Biochemistry Graduate Programs, The Ohio State University, Columbus, Ohio, USA

Amyotrophic lateral sclerosis (ALS) is a atal neurode-generative disease o the motor system. Recent workin rodent models o ALS has shown that insulin-like

growth actor-1 (IGF-1) slows disease progression whendelivered at disease onset. However, IGF-1s mecha-nism o action along the neuromuscular axis remainsunclear. In this study, symptomatic ALS mice receivedIGF-1 through stereotaxic injection o an IGF-1-express-ing viral vector to the deep cerebellar nuclei (DCN),a region o the cerebellum with extensive brain stemand spinal cord connections. We ound that deliveryo IGF-1 to the central nervous system (CNS) reduced

ALS neuropathology, improved muscle strength, andsignicantly extended lie span in ALS mice. To explorethe mechanism o action o IGF-1, we used a newly

developed in vitro model o ALS. We demonstratethat IGF-1 is potently neuroprotective and attenuatesglial cellmediated release o tumor necrosis actor-(TNF-) and nitric oxide (NO). Our results show thatdelivering IGF-1 to the CNS is sucient to delay dis-ease progression in a mouse model o amilial ALS anddemonstrate or the rst time that IGF-1 attenuates thepathological activity o non-neuronal cells that contrib-ute to disease progression. Our ndings highlight aninnovative approach or delivering IGF-1 to the CNS.

Received 2 January 2008; accepted 3 March 2008; published online1 April 2008. doi:10.1038/mt.2008.60

IntroductIonAmyotrophic lateral sclerosis (ALS) is a atal neurodegenerative

disease characterized by a loss o motor neurons in the motor cor-tex, brain stem, and spinal cord. Approximately 20% o diagnosedamilial cases o ALS are due to dominantly inherited mutationsin superoxide dismutase-1(SOD1).1 ransgenic mice that express

the mutant human SOD1 protein recapitulate many pathologicaleatures o ALS and are currently the best available animal modelto study the disease.2

rophic actors, such as insulin-like growth actor-1 (IGF-1),have potent eects on motor neuron survival and have been inves-tigated extensively as potential treatments or ALS.35 Recently, it

has been shown that simultaneous delivery o IGF-1 to the neuro-muscular junction, muscle, and spinal cord by intramuscular injec-tion o an IGF-1-expressing viral vector leads to extended survival

in SOD1G93A mice.6 Although it is evident rom this experimentthat IGF-1 is benecial, it is dicult to conclude which componento the neuromuscular axis IGF-1 is primarily acting on to delay

disease progression. Interestingly, existing evidence suggests thatmuscle may be the principle target o IGF-1 as double transgenicSOD1G93A and MLC/mIg-1 mice show improved survival.7

Te mechanism by which IGF-1 slows disease progression when

delivered at the time o disease onset remains unclear. It is likely thatIGF-1 is delaying motor neuron cell death through the stimulationo antiapoptotic pathways. However, it is also possible that IGF-1

may be attenuating the pathological activity o non-neuronal cells(i.e., astrocytes and microglia) that have been reported to modulateboth disease onset and progression in ALS mice.8,9

In this study, we report that central nervous system (CNS)-restricted delivery o IGF-1 is sucient to modiy disease progressionin ALS mice. We demonstrate that delivery o AAV-IGF-1 vectors

to the deep cerebellar nuclei (DCN) o symptomatic SOD1G93A miceresulted in axonal transport o vector and/or expressed IGF-1 proteinthroughout the brain stem and spinal cord.1014 Concomitant with

IGF-1 expression within the CNS was a proound reduction in neu-ropathology throughout the CNS, increased motor neuron survival,improved motor unction, and a signicant extension o lie span. In

addition, the results o our in vitro studies indicate that IGF-1 maybe delaying disease progression through attenuation o glial cellmediated release o actors [i.e., tumor necrosis actor- (NF-) and

nitric oxide (NO)] known to initiate motor neuron cell death.

resultsdiibi IGF-1 i cns a amiiai AAV-IGF-1 dcnFigure 1a illustrates the connections between the DCN and thespinal cord. Te medial and interposed nuclei receive input rom

Correspondence:James C. Dodge, Genzyme Corporation, 1 Mountain Road, Framingham, Massachusetts 01701-9322, USA.E-mail:[email protected]
http://www.nature.com/doifinder/10.1038/mt.2008.60mailto:[email protected]:[email protected]://www.nature.com/doifinder/10.1038/mt.2008.60mailto:[email protected]


2/9

The American Society o Gene TherapyDCN Delivery o IGF-1 to the Spinal Cord in ALS

Molecular Terapyvol.16no.6june2008 1057

each division o the spinal cord whereas the lateral nucleus receivesinput primarily rom the thoracic division.10,12,13,15,16 All o the cere-bellar nuclei have been reported to send input to the cervical divi-

sion o the spinal cord.14 o determine the potential or targetingmultiple regions o the CNS via the aerent and eerent projec-tion pathways o the DCN, we tested two adeno-associated virus

(AAV) serotypes, AAV2 and AAV1. AAV2 was chosen becausemost clinical studies to date have used this serotype vector. AAV1was selected because it has been previously demonstrated toexpress high levels o transgenes in the brain.17,18 We stereotaxi-

cally injected 2 1010 DNase resistant particles (DRP) o AAV1-IGF-1 into the DCN o 90-day-old SOD1G93A mice and evaluatedIGF-1 expression 20 days aer injection. Positive IGF-1 signal was

observed throughout the hindbrain, brain stem, and spinal cordaer bilateral delivery o the IGF-1-expressing AAV vectors to theDCN. Positive IGF-1 staining was detected in the cerebellar cor-

tex, brain stem (i.e., pontine nucleus, acial nucleus, locus ceru-leus, and vestibular nuclei), and spinal cord. Within the spinal cord(Figure 1b), positive IGF-1 staining was most widely distributed

within the cervical and thoracic divisions with detectable expres-sion ound in the lumbar and sacral regions, demonstrating thatIGF-1 was expressed in all regions o the spinal cord at levels that

may provide trophic support to motor neurons.

divy AAV-IGF-1 dcn i m pi a ia i paAAV1-IGF-1 and AAV2-IGF-1 were tested or their ability to enhance

motor neuron survival in SOD1G93A mice compared with controlAAV1-GFP and AAV2-GFP when injected at disease onset (8890days old). All regions o the spinal cord were evaluated at 110 days

o age or the number o ChA positive cells. AAV1-IGF-1-treatedanimals (17.86 1.91) showed a signicant (P< 0.01) preservationo motor neurons in the cervical region o the spinal cord comparedwith AAV2-IGF-1-treated mice (11.8 1.83) or AAV-GFP-treated

controls (12.74 1.08) (Figure 2a). Both AAV1-IGF-1- (19.96 0.39) and AAV2-IGF-1-treated animals (15.94 1.21) displayed sig-nicantly (P< 0.01) higher numbers o motor neurons per section

compared with AAV-GFP-treated animals (11.74 0.762) in thelumbar region o the spinal cord (Figure 2c). Tere was no dierencein the mean numbers o ChA-positive cells between AAV1-IGF-1-

and AAV-GFP-treated animals in the thoracic or sacral regions othe spinal cord at this time point (Figure 2bandd).

In a separate cohort o animals, survival was assessed by

KaplanMeier survival curves (Figure 2e). AAV-IGF-1 deliveredto the DCN resulted in an ~14-day increase in median survivalcompared with AAV-GFP-treated animals (n = 25 animals/group,

2 = 17.16, P= 0.0007). Median survival o AAV1-IGF-1-treated ani-mals was similar to AAV2-IGF-1-treated animals (133.5 days versus

Cervical

a

b Cervical

AAV1-IGF-1

AAV1-GFP

Thoracic Lumbar Sacral

DCN

Thoracic

Lumbar

SacralMedial

Interposed

Lateral

Syringe

Fig 1 divy via v apab axa ap i ag ivy g pia . (a) Diagramillustrating aerent and eerent connections between the deep cer-ebellar nuclei (DCN) and spinal cord. The DCN is composed o threeseparate nuclei: the lateral (orange), interposed (purple), and medial(yellow). The medial and interposed nuclei receive input rom everyregion (i.e., cervical, thoracic, lumbar, and sacral) o the spinal cordwhereas the lateral receives input only rom the thoracic division. Allthe three nuclei send projections to the cervical division o the spinalcord. Bilateral stereotaxic injections o viral vectors were made betweenthe medial and interposed nuclei. (b) Insulin-like growth actor-1(IGF-1) staining in AAV-GFP- and AAV-IGF-1-treated mice throughouteach segment o the spinal cord. AAV, adeno-associated virus; GFP,green fuorescent protein. This gure is available in color in the onlineversion o the article.

20a b

c

e

d

Motorneurons

persection

Motorneurons

persection

Motorneurons

persection

Motorneurons

persection15

10

5

0AAV1

IGF-1

AAV2

IGF-1

Cervical Thoracic

LumbarSacral

AAV

GFP

WT

AAV1IGF-1

AAV2IGF-1

AAVGFP

WT

AAV1

IGF-1

AAV2

IGF-1

AAV

GFP

WT

AAV1IGF-1

AAV2IGF-1

AAVGFP

WT

15

10

5

0

15

10

5

0

20

15

10

5

0

100AAV1-IGF-1

AAV2-IGF-1

AAV1-GFP

AAV2-GFP

Percentsurvival 75

50

25

0

110 120 130

Age of death (days)

140 150 160

*

***

*

Fig 2 AAV-IGF-1-pm m viva a aya i amypi aa i mi. Motor neuron counts in(a) cervical, (b) thoracic, (c) lumbar, and (d) sacral regions o the spinalcord. KaplanMeier survival analysis o AAV1-IGF-1-, AAV2-IGF-1-, AAV1-GFP-, and AAV2-GFP-treated animals (e). Mice were scored as deadwhen they could no longer right themselves within 30 seconds o beingplaced on their back. Green fuorescent protein (GFP)-treated mice areindicated in green and insulin-like growth actor-1 (IGF-1)-treated miceare indicated in red. AAV, adeno-associated virus; WT, wild type.


3/9


1058 www.moleculartherapy.org vol.16no.6june2008

134 days, respectively) and the median survival o AAV1-GFP- andAAV2-GFP-treated animals was comparable (121.5 days versus120 days). Tere was no dierence in survival between untreated

controls and AAV-GFP-treated animals (data not shown).

Fia bf dcn ivy AAV-IGF-1A battery o motor unction tests was used to monitor disease pro-gression. Forelimb grip strength measurements demonstrated thatAAV-IGF-1-treated animals maintained statistically (P < 0.05)greater grip strength rom 103 days o age through 131 days o

age compared with those administered AAV-GFP (Figure 3a). Inaddition, animals treated with IGF-1 showed remarkable, statis-tically signicant (P< 0.05) increases in hindlimb grip strength

(Figure 3b). Rotarod tests also demonstrated that AAV-IGF-1-treated animals maintained their ability to coordinate their move-ment or a longer period than AAV-GFP-treated animals rom

110 days o age until end stage (P < 0.05 rom 110 days o ageonward, n = 25 animals/group). In all o the motor unction tests,there were no statistical dierences observed between animals

treated with AAV1-IGF-1 and AAV2-IGF-1 other than one timepoint at 124 days o age in the rotarod test (Figure 3c).

IGF-1 a g bai a pia AAV-IGF-1-a mio determine whether IGF-1 was expressed at similar levels by the

two serotype vectors, we measured the levels o the trophic actorusing an enzyme-linked immunosorbent assay that recognizedthe expressed human IGF-1 and not the endogenous murine

counterpart. Detectable levels o human IGF-1 were noted inall o the regions o the brain and spinal cord o mice injectedwith AAV-IGF-1. Highest levels were ound in the cerebellumand cervical region o the spinal cord, which were at or near the

site o injection with no statistical dierences between AAV1 andAAV2 (Figure 4a). No IGF-1 was detected in the serum o AAV-IGF-1-treated animals, indicating that the IGF-1 delivery was

not systemic. Little to no IGF-1 was detected in green fuorescentprotein (GFP)-treated animals, with background levels detected


4/9



o the spinal cord (Figure 4b). No IGF-1 mRNA was ound eitherin the AAV1-GFP-treated (Figure 4b) or AAV2-GFP-treatedanimals (data not shown), or in the reverse transcriptaseminus

controls. Tese results demonstrate that both the AAV1-IGF-1and AAV2-IGF-1 vectors underwent retrograde transport to allregions o the spinal cord aer DCN injection.

IGF-1-xpig AAV v Als-aiapagy i sod1G93A miWe next evaluated the ability o this therapy to attenuate the neu-

ropathological eatures characteristic o ALS disease. Activatedmicroglia and astrocytes contribute to the propagation o the dis-ease process in ALS.4 Widespread gliosis is readily apparent in the

brain stem and spinal cord o both human ALS patients and mousemodels o the disease.19,20 In addition, biochemical assays and geneexpression proling studies showed that infammatory cascades

are activated beore and during motor neuron degeneration.21,22Microglial activation, astrogliosis, NO synthase expression, andperoxynitrite levels were assessed in mice treated with the AAV-

IGF-1 and AAV-GFP vectors. MetaMorph analysis o our resultsshowed that delivering AAV-IGF-1 to the DCN led to a reductionin gliosis both within the brain stem and throughout the spinal

cord at 110 days o age compared with AAV-GFP-treated animals.Markers o microglial activation (F4/80 staining) and astrogliosis(glial brillary acidic protein staining) were diminished throughout

the brain stem, including the motor trigeminal, hypoglossal, and

acial nuclei (Figures 5a and cand6a andc). Troughout the entirelength o the spinal cord, microglial activation and astrogliosis werealso dramatically reduced (Figures 5b and cand6b andc).

NO has been implicated as a contributing actor to the patho-genesis o ALS.23 Upregulation o NO has been shown to be involvedin initiating Fas-triggered cell death, a programmed cell death path-

way that appears to be restricted to motor neurons.24 Elevated NOhas also been linked to the generation o peroxynitrite, ormed bythe reaction o NO with superoxide anions, resulting in the nitra-tion o tyrosine residues in neurolaments. Tis, in turn, causes

irreversible inhibition o the mitochondrial respiratory chain, andinhibition o glutamate transporter activity.25 Moreover, increased3-nitrotyrosine immunoreactivity (a marker o peroxynitrite) has

been reported in the spinal cord o both sporadic and amilial ALSpatients.26 Similar elevations in 3-nitrotyrosine have also beenobserved in the CNS o ALS mouse models.27,28 MetaMorph analysis

o our results showed that delivery o IGF-1 resulted in reductionsin the levels o both NO synthase (Figure 7a and c) and 3-nitroty-rosine (Figure 7bandc) throughout the spinal cord.

IGF-1 i piv i m Alsa a iibi migia aivai aagia xiiyRecent studies using embryonic stem cellderived motor neuronshave demonstrated that in vitro models o ALS could be developed

a

b

c

AAV-GFP

AAV-IGF-1

AAV-GFP

AAV-IGF-1

AAV-GFP

AAV-IGF-1

WT AAV-GFP

AAV-IGF-1

WT

*

7

Cervical Thoracic Lumbar Sacral

12 Mo5

3.0 1007

F4/80 staining in brain stem F4/80 staining inlumbar spinal cord

Integratedintensity

Integratedintensity 2.0 10

07

1.5 1007

1.0 1007

0.5 1007

0.00

2.0 1007

1.0 1007

0.0

Fig 5 AAV-IGF-1 aa migia aivai i amypiaa i (Als) mi. Microglial activation (F4/80 staining) in the(a) brain stem (7 = acial nucleus, 12 = hypoglossal nucleus, and Mo5 =motor trigeminal nucleus) and (b) spinal cord in 110-day-old superoxidedismutase-1 mice that were treated with either AAV-GFP or AAV-IGF-1 at90 days o age. (c) MetaMorph analysis o F4/80-stained brain stem andspinal cord sections taken rom ALS mice treated with either AAV-IGF-1or AAV-GFP and wild-type (WT) controls. AAV, adeno-associated virus;GFP, green fuorescent protein; IGF, insulin-like growth actor.

a

b

c

AAV-GFP

A

AV-IGF-1

AAV-GFP

AAV-IGF-1

AAV-GFP

AAV-IGF-1

WT AAV-GFP

AAV-IGF-1

*** *

WT

7


12 Mo5

3.0 1007

GFAP staining in brain stem GFAP staining inlumbar spinal cord

Integrate

dintensity

Integrate

dintensity 5.0 10

07

4.0 1007

3.0 1007

1.0 1007

2.0 1007

0.0

2.0 1007

1.0 1007

0.0

Fig 6 AAV-IGF-1 igifay aa agii i amy-pi aa i (Als) mi.Astrogliosis [glial brillary acidic pro-tein (GFAP) staining] in the (a) brain stem (7 = acial nucleus, 12 =hypoglossal nucleus, and Mo5 = motor trigeminal nucleus) and (b) spinalcord in 110 day old superoxide dismutase-1 mice that were treated witheither AAV-GFP or AAV-IGF-1 at 90 days o age. (c) MetaMorph analysiso GFAP-stained brain stem and spinal cord sections taken rom ALS micetreated with either AAV-IGF-1 or AAV-GFP (P< 0.05) and wild-type (WT)controls. AAV, adeno-associated virus; GFP, green fuorescent protein;IGF, insulin-like growth actor.


5/9


1060 www.moleculartherapy.org vol.16no.6june2008

that mimic the motor neuron death seen in animal models o the

disease.29,30 We developed similar models using embryonic stemcellderived motor neurons containing the Hb9-GFP reporterthat were transduced with a lentivirus containing the SOD1G93A

gene or control SOD1W. As previously shown, the mutationhad minimal eects when expressed only in motor neurons.29,30However, when motor neurons with or without the SOD1G93A gene

were cocultured with astrocytes containing the SOD1G93A, motorneurons exhibited shorter axon lengths, increased cell death, andapoptosis shown by caspase-9 activation, which demonstrates and

conrms that astrocytes expressing the mutant SOD1 are toxic tomotor neurons (Figure 8a and b). o test whether IGF-1 could res-cue the motor neuron toxicity, we replaced the medium rom the

SOD1G93A

-expressing cocultures with either IGF-1-conditionedmedium or mock-transected control-conditioned medium andcompared the results with wild-type cocultures. We observed sig-

nicant rescue and neuroprotective eects o IGF-1 in cocultureexperiments with motor neurons and astrocytes both containingthe SOD1G93A mutation, which was comparable to control wild-type cultures. IGF-1 treatment resulted in extensive preservation

o neuritic extensions along with decreased caspase-9 activationindicating that IGF-1 was potently neuroprotective in this model(Figure 8a and b). We next sought to determine whether IGF-1

may be acting to delay glial cell activation, because earlier stud-ies have demonstrated that glial cells containing the SOD1G93Amutation were a major contributor to disease progression and

motor neuron death. We obtained the BV2 microglial cell lineand transduced the cells using a lentivirus expressing wild-typeSOD1 or SOD1G93A. Upon lipopolysaccharide induction, these

cells produce high levels o NF- and NO. SOD1G93A microgliaexpressed higher levels o both NF- and NO compared withwild-type SOD1 microglia as previously demonstrated, suggest-

ing that SOD1G93A mutation modestly activated these microg-lia.31,32 Consistent with our earlier in vivo experiments, when BV2microglial cells were cultured with IGF-1-conditioned mediabeore lipopolysaccharide-mediated activation, IGF-1 signicantly

reduced NF- levels and completely suppressed the NO releaseto baseline levels o nonstimulated microglia, suggesting thatIGF-1 directly attenuates microglial cell activation (Figure 8c).

We next tested the specic action o IGF-1 in our ALS-motorneuron/astrocyte coculture system using wild-type motor neu-rons or SOD1G93A motor neurons cocultured with SOD1G93A astro-

cytes. Because IGF-1 is a secreted molecule, it is dicult to testthe eects o IGF-1 protein solely on motor neurons or astrocytes;hence, we utilized a signaling pathway o IGF-1 to test our hypoth-

esis that IGF-1 was acting both on motor neurons and astrocytesor neuroprotection. IGF-1 is one o the most potent natural acti- vators o the AK signaling pathway. We conrmed that IGF-1

could activate AK in astrocytes (data not shown) and thereoreused an adenovirus encoding a constitutively activated AK thatwas restricted to expression solely in astrocytes to mimic IGF-1

signaling. A dominant negative AK adenovirus expressed onlyin astrocytes was also used as a negative control and as a controlto inhibit IGF-1 signaling through AK activation in astrocytes.As demonstrated in our earlier study, motor neurons with or with-

out SOD1G93A perished when cultured on SOD1G93A astrocytes.IGF-1 added to the cultures signicantly rescued the motor neu-rons rom this toxicity. Interestingly, when motor neurons were

cultured on top o SOD1G93A astrocytes expressing the constitu-tively activated AK, there was signicant protection o motorneurons compared with untreated (Figure 8d) or dominant neg-

ative AK only expressing astrocytes (data not shown). o testwhether IGF-1 signaling was required in astrocytes or motorneuron protection, a dominant negative AK was expressed in

astrocytes and IGF-1-conditioned media were added to the cocul-ture. Blocking AK signaling in astrocytes signicantly reducedmotor neuron survival, but did not completely abolish the neu-

roprotective eects o IGF-1, indicating that IGF-1 signaling toactivate AK acts on both motor neurons and astrocytes. Teseresults suggest that IGF-1 signaling via AK activation in astro-

cytes is sucient in part to provide protection to motor neuronsrom astrocyte-derived toxicity and that there are additive eectso motor neuron protection by IGF-1 when both motor neurons

and astroctyes are exposed to IGF-1.

dIscussIonrophic actors such as IGF-1 have shown promise or the treat-

ment o ALS.35 In this study, we report that CNS-restricteddelivery o IGF-1 is sucient to modiy disease progression insymptomatic ALS mice. Specically, we showed that injecting

a recombinant AAV vector encoding IGF-1 within the DCNo SOD1G93A mice resulted in axonal transport o vector and/orexpressed IGF-1 protein to the brain stem and all segments o the

AAV-GFP

AAV-IGF-1

AAV-GFP

AAV-IGF-1



a

b

AAV-GFP

AAV-IGF-1

WT AAV-GFP

AAV-IGF-1

WT

Nitrotyrosine staining inlumbar spinal cord

NOS staining inlumbar spinal cord

In

tegratedintensity

In

tegratedintensity

5.0 1007

6.0 1007

4.0 1007

3.0 1007

1.0 1007

2.0 1007

0.0

1.0 1007

1.5 1007

0.5 1007

0.00

c

**

*

Fig 7 AAV-IGF-1 am ia-i vaii ii xi ya (nos) aiviy a pxyii maii amypi aa i (Als) mi. (a) NOS and (b) 3-nitro-tyrosine staining (peroxynitrite marker) in the spinal cord o 110-day-oldsuperoxide dismutase-1 mice treated with either AAV-GFP or AAV-IGF-1at 90 days o age. (c) MetaMorph analysis o NOS and 3-nitrotyrosine-stained spinal cord sections taken rom ALS mice treated with either AAV-IGF-1 or AAV-GFP (P < 0.05) and wild-type (WT) controls. AAV,adeno-associated virus; GFP, green fuorescent protein; IGF, insulin-likegrowth actor.


6/9



spinal cord. Tis, in turn, led to improved muscle unction and a

signicant extension o lie span. Furthermore, IGF-1 also attenu-ated astrogliosis, microglial activation, peroxynitrite ormation,and glial cellmediated release o NF- and NO.

Results obtained using mouse models o motor neuron diseasehave demonstrated that trophic actors (e.g., IGF-1, BDNF, CNF,and GDNF) have potent eects on motor neuron survival.35

However, systemic administration o some o these recombinanttrophic actors into subjects with ALS showed only verymodest clinical benet.3336 Studies in ALS mice suggested that

inadequate delivery o these trophic growth actors to the CNSmay have been responsible or the poor response. Only systemicadministration o vascular endothelial growth actor has been

reported to be eective in treating SOD1G93A mice.37 Intrathecaladministration o puried IGF-1 to the same mouse model wasalso ecacious.38 However, in both cases, positive eects werereported only when treatment was initiated in presymptomatic

animals. In contrast to the results observed with systemic orintrathecal delivery o puried trophic actors, intramuscularinjections o viral vectors encoding these actors demonstrated

signicant therapeutic benet in the SOD1G93A mice, even whenadministered aer the onset o overt disease symptoms.6,39 Teresults o this study indicate that trophic actor delivery to the

CNS is sucient to modiy disease progression in symptomatic

ALS mice. An advantage o this delivery strategy over existingapproaches is that it permits the targeting o multiple areas thatundergo neurodegeneration in ALS with a single injection site

and obviates the need or injecting directly into the spinal cordwhere neurodegeneration is taking place. Comparison o sur-vival benets achieved with DCN versus intramuscular delivery

o AAV-IGF-1 is dicult, given that a death event in an ALSmouse is articially determined (i.e., occurs when the mouse canno longer right itsel within 30 seconds). In the mouse model,

testing a therapeutic is limited to measuring the ability to oerprotection to motor neurons predominantly residing in thelumbar division o the spinal cord, which is responsible or the

righting refex. Indeed direct intraspinal injections o AAV-IGF-1led to signicant increases in survival.40 However, given thatrespiratory ailure is the primary cause o death in ALS patients,we believe that delivering AAV-IGF-1 to the DCN may oer an

advantage in that it permits targeting regions o the CNS thatcontrol respiration. Tis, in turn, may lead to a level o ecacy inALS patients beyond what is observed in ALS mice.

Cellular mechanisms that modulate disease progression inALS have not been known until just recently. While disease onsetis initiated by motor neurons in ALS, it appears that glial cells

HB9-GFPa b c

d e

SOD1-G93A

SOD1

-G93A+

IG

F-1

Wildtype

60*

*

*#30

%Day0HB9-GFP+

cellsremaining

0

60

30

%Day0HB9-GFP+

cellsremaining

0IGF-1

(media)

Astrocytes

dNAKT

cAAKT

WT SOD1-G93A

WT motor neurons

Motor neurons

GFAP Merged

100

%Day0HB9-GFP+

cellsremaining

TNF-(

pg/ml)

Cl.caspase9+

cells/EB

50

00 1

Day4

+

+ +

SOD1-G93A WT

**

6

4

2

0IGF-1

SOD1-WTSOD1-G93A + IGF-1SOD1-G93A

2,000

*

*

*

*

1,500

1,000

500

0LPS

IGF-1+ + + +

+

Wild type SOD1-G93A

+

Nitrite+

nitrate(mol/l) 30

**

** *

*

20

10

0LPS

IGF-1

+ + + +

+

Wild type SOD1-G93A

+

IGF-1(media)

Astrocytes

dNAKT

cAAKT

WT SOD1-G93A

+ +

WT SOD1-G93A

*

* *

#

IGF-1

Nit ric ox ide TNF-

Glutamate

Calcium

Superoxide

Peroxynitrate

Motor neuroncell death

Microglial activationastrogliosis

+

+ +

AKT- p

Fig 8 Ii-i g a-1 (IGF-1) amypi aa i (Als) m xiiy a aa gia aivaia xiiy i a in vitro m Als. (a) SOD1-G93A motor neurons cultured with SOD1-G93A astrocytes in the presence o IGF-1extend axons comparable to wild-type (WT) motor neurons cocultured with WT astrocytes. (b) SOD1-G93A motor neurons in a coculture with SOD1-G93A astrocytes survive longer with IGF-1 assessed by HB9-GFP counts and cleaved (cl.) caspase-9 + cells per embryoid body (EB) and compared toSOD1-WT motor neurons cultured with WT astrocytes. (c) SOD1-G93A microglia produce increased amounts o tumor necrosis actor- (TNF-) andnitric oxide and IGF-1 attenuates the release o these actors. (d) Coculture o WT or SOD1-G93A motor neurons with WT or SOD1-G93A containingastroctyes in the presence o IGF-1-conditioned media and/or astrocytes expressing dominant negative (dN) AKT or constitutively active (cA) AKTdemonstrates IGF-1s neuroprotective ability and its actions on both motor neurons and astrocytes to protect motor neuron survival. (e) IGF-1 servesdual roles as an antiapoptotic actor and to block microglial activation and astrogliosis or motor neuron protection in ALS via activation o AKT. (*P

DODGE (2008) Delivery of AAV-IGF-1 to the CNS Extends

Documents

Transcript of DODGE (2008) Delivery of AAV-IGF-1 to the CNS Extends