Dissen Progress in siRNA Final ppt prese

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Presented by Dr. Gregory Dissen at the Alliance for Contraception in Cats & Dogs’ 4th International Symposium on Non-Surgical Contraceptive Methods of Pet Population Control, April 8-10, 2010, in Dallas, Texas, U.S.

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Slide 1: 

Progress in siRNA Targeting in the Central Nervous System: Where are we on Proof of Principle? Gregory A. Dissen, Jodi McBride, Alejandro Lomniczi, Beverly Davidson, and Sergio R. Ojeda

Slide 2: 

Outline Present use of RNA interference to study reproduction Potential viral Delivery Methods Viral vector that has the ability to cross the Blood Brain Barrier Target a viral vector to an area of the CNS

Slide 3: 

Round worms RNA interference was discovered in plants and worms Petunias siRNAs act through a process known as RNA interference RNA interference occurs throughout phylogeny RNA interference is active in all plants and animals

Slide 4: 

Target to induce Infertility in the CNS: Hypothalamus Releases GnRH Portal vessels of the Medium Eminence GnRH acts on The Pituitary Gonadotropins Gonads Ovary Testes

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Problem: Blood Brain Barrier BBB System of tight Junctions Between Endothelial cells Supported by Astrocytes Restricts access to the neurons of the CNS Objective: Deliver the siRNAs across the BBB to hypothalamic neurons Target: Hypothalamus

Slide 6: 

Tiemann and Rossi EMBO Mol Med 2009 1:142-151 siRNA Delivery Methods: Viral Non-viral

Slide 7: 

Viral mediated siRNA Delivery Lentivirus Vectors Adeno-associated virus Vectors Kim and Rossi Nature Reviews Genetics 8, 173–184 (March 2007) | doi:10.1038/nrg2006 If the virus has access to hypothalamic neurons, will infection and delivery of siRNA occur?

Slide 8: 

Viral mediated siRNA Delivery Lentivirus Vectors Infect proliferating and non-proliferating cells Not subject to ‘shut-off’ like other retroviruses Genomic insertion of the proviral DNA has less potential for insertional oncogenesis than other retroviruses Minimal inflammatory response in the brain no cytotoxicity

Slide 9: 

Y stranded Verma and Somia 1997 Cytoplasm Life cycle of the Lentiviral Vector: Packaging Infection Reverse Transcription Integration Transcription siRNA

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Packaging Transgene Envelope Lentiviral Vector System Transfection pLV pMDLpg.RRE pRSV-Rev pMD.G Transfected 293T cells CaPO4 CaPO4

Slide 11: 

Identification of genes related to puberty using microarrays Targets If we interfere with the appropriate Target in the CNS can we block fertility?

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GenBank ID Gene Name R44477 ESTs AF181849 inhibitor of growth 1 family, member 1 AW970315 hypothetical protein FLJ20514 AW444959 KIAA0500 protein AI376143 chromosome 14 open reading frame 4 BE786886 FKBP-associated protein AI564701 major histocompatibility complex, class I, C U79275 hypothetical protein AB018278 KIAA0735 gene product; synaptic vesicle protein 2B homolog D45352 superiorcervical ganglia, neural specific 10 AA505399 acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain NM_001659 ADP-ribosylation factor 3 AI439625 KIAA0618 gene product BE266136 hypothetical protein FLJ20113 AK024695 Homo sapiens cDNA: FLJ21042 fis, clone CAE11204 NM_003866 inositol polyphosphate-4-phosphatase, type II, 105kD NM_003613 cartilage intermediate layer protein, nucleotide pyrophosphohydrolase AA173284 myosin X U63810 WD40 protein Ciao1 AW675602 splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing factor) AF086713 RAS protein activator like 1 (GAP1 like) AW958325 fetal hypothetical protein AW631118 fatty acid binding protein 4, adipocyte U60521 caspase 9, apoptosis-related cysteine protease AI619701 ESTs AF017445 fuctose-1-phosphate guanylyltransferase BE311461 neuroblastoma, suppression of tumorigenicity 1 Genes with Continued Increased Expression 0.0 0.5 1.0 1.5 2.0 2.5 3.0 1-2 years 2- 3 years 3- 4 years Fold Change Chromosome 14 Open Reading Frame 4 EST (R44477) Juvenile to early Puberty Early to Midpuberty C14ORF4, a gene of unknown function Enhanced at Puberty EAP1 Example:

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EAP-1 is a nuclear protein expressed in several neuronal subsets, including GnRH neurons 20μm 40μm 10μm Red = GnRH neurons Green = EAP-1 Blue = cell nuclei Red = EAP1 Blue = Hoescht Rat – POA GnRH – EAP1 EAP1 Neuron

Slide 14: 

EAP-1 is expressed in GnRH neurons. EAP-1 is also expressed in the regulatory network surrounding GnRH neurons both excitatory (glutamatergic, KiSS1) and inhibitory (gabaergic, enkephalinergic). EAP-1 is localized to the cell nucleus, where it appears to be associated with the nuclear matrix and not with condensed chromatin, suggesting an involvement in transcriptional regulation.

Slide 15: 

EAP-1 has dual repressive and activating transcriptional activities, which are both cell and promoter-specific. EAP-1 is able regulate the transcriptional activity of genes involved in the control of the LHRH neuronal network, including LHRH itself. Does a loss of hypothalamic EAP-1 expression have an impact on reproductive function?

Slide 16: 

5’LTR cPPT Promoter Reporter Gene wPRE SIN3’LTR RRE Lentiviral Vector System (3rd generation) SD Y 486 bp Insertion of shRNA expression Cassette shRNA EAP1 specific

Slide 17: 

Questions? Will the virus infect hypothalamic neurons if the blood brain barrier is bypassed? Will the shRNA/siRNA suppress the target in the hypothalamus? Will suppression of the target (EAP1) result in infertility?

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Injection of Lentivirus in the rat brain Target: cell bodies of GnRH neurons Located in the area known as the Anteroventral periventricular nucleus AVPV

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V OC AVPV Green: GFP of LV-EAP1 siRNA Blue: Hoescht Injection of Lentivirus in the rat brain

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GFP positive cells Most: Neuronal morphology

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eGFP = green LHRH = red Example: GFP = Green LV-EAP1 siRNA Co-localized with GnRH neuron (Red)

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Infected (GFP) = green EAP1 = red

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Infected (GFP) = green EAP1 = red Reduced EAP1 (red) in infected cells

Slide 24: 

Question? Will the virus infect hypothalamic neurons if the blood brain barrier is bypassed? Will the shRNA/siRNA suppress the target in the hypothalamus? Will suppression of the target (EAP1) result in infertility? YES YES

Slide 25: 

Knocking down EAP-1 gene expression in the POA delays the onset of puberty in female rats LV EAP-1 hp siRNA Age at first ovulation (days) (9) (10) *

Slide 26: 

The rat has a 4 day estrous cycle: Proestrus – Estrus – Diestrus 1 – Diestrus 2 P E D1 D2 Age (days) Days of the Cycle are plotted to give visual appraisal of cyclicity

Slide 27: 

Knocking down rat EAP-1 gene expression in the POA disrupts estrous cyclicity LV-control EAP-1 shRNA P E D1 D2 P E D1 D2 P E D1 D2 P E D1 D2 P E D1 D2 P E D1 D2 Age (days)

Slide 28: 

Question? Will the virus infect hypothalamic neurons if the blood brain barrier is bypassed? Will the shRNA/siRNA suppress the target in the hypothalamus? Will suppression of the target (EAP1) result in infertility? YES YES Journal of Clinical Investigation 117:2145; 2007 YES

Slide 29: 

Question? Will the virus infect hypothalamic neurons if the blood brain barrier is bypassed? Will the shRNA/siRNA suppress the target in the hypothalamus? Will suppression of the target (EAP1) result in infertility? Will suppression of EAP1 result in infertility in a Species other than rodents? YES YES YES

Slide 30: 

Problem with shRNAs 49 shRNA vectors: 36 resulted in dose-dependent liver injury 23 causing death

Slide 31: 

microRNAs vs shRNAs Artificial miRNAs: Expressed at lower levels and processed efficiently Expression well tolerated miRNA vectors better suited for therapeutic silencing in the Mammalian brain

Slide 32: 

Clone Xho I – EcoRI 110 bp Stegmeier, 2005 Artificial miRNA Replaceable Hairpin structure

Slide 33: 

Injection of Lentivirus in the monkey brain Target: cell bodies of GnRH neurons Located in the area known as the arcuate nucleus

Slide 34: 

Arcuate nucleus Median Eminence Green = GFP Lentivirus 3V

Slide 35: 

Green = GFP Red = GFAP Blue = Hoescht

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GnRH GFP Merge Kisspeptin GFP Merge Virus expressed in GnRH And Kisspeptin neurons

Slide 37: 

Monkeys = Menstrual Cycle (28 days)

Slide 38: 

Control Virus EAP1 siRNA Virus

Slide 39: 

Question? Will the virus infect hypothalamic neurons if the blood brain barrier is bypassed? Will the shRNA/siRNA suppress the target in the hypothalamus? Will suppression of the target (EAP1) result in infertility? Will suppression of EAP1 result in infertility in a Species other than rodents? YES YES YES YES

Slide 40: 

Past the BBB Inhibition of target (EAP1) expression: Infertility Rats, Nonhuman primates siRNAs Delivered to the hypothalamic neurons with Lentivirus Problem: Administration – stereotaxic surgery is not an option Lentivirus pseudotyped with VSV-G cannot be Administered systemically Adeno-associated virus can be administered systemically

Slide 41: 

Viral mediated siRNA Delivery Adeno-associated virus Vectors Can be administered systemically/intravenously Infect proliferating and non-proliferating cells Primarily will not integrate into the genome Maintained as an episome in non-proliferating cells Minimal immune response

Slide 42: 

AAV Productive Infection Virus binding to Receptor Endocytosis by Host Cell Endosomal trafficking Escape from the Endosome Virion Uncoating Entry into the Nucleus Single to Double stranded Permanent Episome Coura and Nardi 2008

Slide 43: 

Primate AAVs Gao 2004 Many varieties of AAVs Known as: Serotypes Serotype is Determined by The Capsid Protein Serotype Determines Tropism of the Virus: What cells/tissues Are infected

Slide 44: 

E2A E4 VA RNA Rep Capsid E1 Transgene ITR Vector siRNA AAV Vector System Co-transfection 3 plasmids Special 293T cells Serotype Determined By Capsid Protein

Slide 45: 

AAV genome plasmid Packaging plasmid Virion Target Tissue www.tigem.it Reporter/siRNA ITR 2 ITR 2 Muscle, CNS Muscle, Liver, Spleen, CNS, Retina Inner ear, Liver CNS, Retina Lung, CNS Muscle, Liver, Retina Liver, Muscle, Pancreas, CNS Lung, Liver, Muscle, CNS Pancreas, Retina CNS Recombinant AAVs Genomic Backbone Serotype Specificity

Slide 46: 

Scheme for AAVmiRNA injections and analyses N171-82Q transgenic mice and WT littermates, 5 weeks of age Q-PCR Immunohistochemistry Dose injected per hemisphere: 2.5e10 vg Tg-AAV-miRNA 2.4-GFP Tg-AAV-GFP Tg-Buffer control (FB) Wildtype Buffer control Euthanized 20 weeks post-surgery If bypass the BBB Will AAV2/1 infect Neurons?

Slide 47: 

Distribution of GFP+ cells following a single injection into striatum Yes – AAV readily infects neurons in the CNS if bypass the BBB - Mouse

Slide 48: 

Cellular phenotype of striatal cells transduced with AAV1miRNA-GFP oligodendrocytes astrocytes neurons Preferential expression In neurons

Slide 49: 

Cortex along the needle tract, AAV1-GFP inj. AAV2/1 clearly infects neurons Monkey

Slide 50: 

AAV1-GFP AAV5-mCHERRY AAV1 and AAV5 demonstrate differing cell tropism Preferentially neuronal Vector serotypes exhibit differing tropism within the brain Preferentially astrocytic If bypass the BBB the AAV will infect cells of the CNS Is there a nonsurgical method to bypass the BBB?

Slide 51: 

Applying a systemic delivery strategy to achieve widespread transduction throughout the CNS: AAV9 AAV9-GFP injection in P1 mouse pups transduced neurons and astrocytes AAV9-GFP injection in adult mice preferentially transduced astrocytes AAV9-GFP injection in adult mice transduced Neurons in the brain and motor neurons in the spinal cord Will we get neuronal transduction with AAV9? IV injection of AAV9-GFP in mice – Transduction?

Slide 52: 

GFP expression throughout the brain following Systemic, venous AAV9-GFP injection AAV9-GFP injection PBS control injection Both sections stained with anti-GFP antibody 3 x 1012 vgs

Slide 53: 

Striatum Cortex Ctx. Str. Endothelial cells Transduced with Virus Neurons Transduced Astrocytes Transduced

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Striatum Cortex Ctx. Str. Medium spiny neuron

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Dentate gyrus CA1-hippocampus DG CA1 Found Neurons Throughout the CNS Transduced by AAV9-GFP

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3rd Ven. Median Eminence Arcuate nucleus Median Eminence is a Circumventricular Organ

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AAV in Peripheral Tissues Following Systemic Administration?

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Interstitial cells of Leydig in testis DAPI GFP 17 -hydroxylase Merge AAV2/9 Transduces Cells Outside the Tubules Testes Blood Barrier

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Ovary DAPI GFP Merge AAV2/9 Primarily Transduces Cells Outside the Follicle

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If Bypass the BBB with lentiviral vectors and deliver siRNA into the neuronal network controlling reproduction = Infertility Proof of Principle: Where are We? AAV vectors will infect neurons if bypass the BBB AAV2/9 vectors will infect neurons within the BBB Especially target neurons located in circumventricular areas Is it possible to target an AAV vector to the hypothalamus? Can the Tropism of a viral vector be modified?

Slide 61: 

AAV Coura and Nardi 2008 Capsid Protein determines serotype and Tropism Alter the capsid to match a specific receptor The Tropism will Determine the Target Cell Rather than picking A target we are “Panning” for a Target

Slide 62: 

Phage Display to Identify Novel Peptides for Targeted Gene Therapy Work et al., 2002 Bio-panning 2009 Panning

Slide 63: 

Arap, 2005 What Is: Phage Display? Library of Random peptide 7-mers Displayed on pIII ofM13 phage Means a peptide is Displayed on the pIII Domain What Are We: Displaying? Why?

Slide 64: 

Bio-panning New England Biolabs Binding Washing Recovery Repeat Convergence

Bio-panning : 

Bio-panning Identify peptides that can bind neurons of the hypothalamus Use phage display biopanning Engineer targeting peptides into AAV2 capsid and produce peptide modified AAV2 (PM-AAVs) Test in vivo Test if peripheral delivery of PM-AAV can bind neurons of the hypothalamus Scheme of Tropism modified AAV2 From Muller et al, Nat Biot 21:1040, 2003

Slide 66: 

Bio-panning Arap, 2005 Injection Binding Washing Recovery Convergence

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Mouse Chen, 2009 Red = AAV infected Green = Neurons Mouse Cortex Integrin Binding

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Deliver siRNA AAV9 Delivery Summary Targeted Virus Permanent Infertility Viral Hypothalamus Infertility Cross BBB Altered Tropism Neuronal Expression CNS Targeting

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Acknowledgements Michelson Grant Davidson and Ojeda National Institutes of Health National Institutes of Child Health and Human Development Ojeda and Dissen Ojeda Lab Davidson Lab

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