Grant Proposal:

Human APP Isoform Splice-Site Polymorphism Targeting Using Mosaic AAV-2 Mediated RNAi
Nick Wisner

Abstract There are strong links between several variants of the amyloid precursor protein (APP) and Alzheimers disease.1 For example, the -amyloid (A) plaques accumulate in individuals with mutations in membrane spanning region of the APP. Cleavage of dysfunctional amyloid proteins from surface receptors in the brain results in the formation of these plaques, that are pathogenically associated with Alzheimers disease. Of these transcript variants, most have a splice site polymorphism that is conserved; lacking an in-frame exon. The aim of this study is to silence the mutant isoforms using RNA interference (RNAi) in the form of short-hairpin RNA (shRNA) produced by a recombinant mosaic adeno-associated virus, serotype 2, vector. The shRNA will include the conserved splice site polymorphism identified from the various isoforms. A rescue experiment will be used as a control to determine whether a change in phenotypic behavior is a direct result of the shRNA. Introduction Alzheimers disease (AD) is a neurodegenerative disorder that occurs in the elderly, and is considered the most common form of dementia. The disease is definitively diagnosed by the presence of extracellular amyloid plaques that coalesce in the brain, post-mortem. The plaques consist of 40 to 42 amino acid peptide chains that are thought to arise from a dysfunctional subunit of the amyloid precursor protein (APP)2. The cleavage of this subunit is catalyzed by and secretases from neuron membranes2. The A subunit is a transmembrane region that is released following this cleavage. 1,2 Because most of the concern with APP is focused on the A region, most researchers are focused only on silencing mRNA that vary from the wild type here. However, other evidence suggests

that polar mutations occur upstream of the A region can augment the proliferation of A plaques.3 Thus, I propose to target a sequence that is conserved over several variants of the APP, but occurs further upstream from the A region. There exists a splice site mutation where an in-frame exon, Exon 3, that is maintained in the wild type, is not present in the greater number of APP variants, (Isoform(s) A,B,C,H,I,J) Fig. 5. These variants have a unique nucleotide sequence that extends from the 3 end of exon 2 into the 5 end of exon 4, exon 3 being the missing in-frame exon; both exons 4 & 3 start unique (Fig. 1). Because the wild type mRNA will not contain this sequence Keywords: Alzheimers Disease, APP, mosaic AAV-2 vector
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RNAi targeting this sequence should not interfere with the wild type mRNA. The goal of this project is to determine if this type of gene targeting will improve on the efficacy of previous RNAi of similar mRNA transcripts, as well as to better the understanding of how these upstream splice site mutations are expressed in the mutant protein and what kinds of effects they can have. A secondary goal of this experiment is to construct a viral vector that can efficiently carryout RNAi in neurons, using cell specific targeting. Because the variants investigated in this experiment contain a mutated region that is located on the cell surface, cell specific targeting using these regions should be possible. The purpose for targeting mutated neurons, over the rest of the central nervous system (CNS), is to lower the dosage needed to result in a healthy phenotype. I intend to construct a mosaic AAV-2 viral vector to express immunoglobulin binding proteins in the viral capsid, that will bind targeting antibodies raised against the mutant protein, and the unique, afore mentioned, sequence4. The reason for using both the mutated protein and the peptide sequence is because it may turn out that the sequence is exposed to immune cells, and having a mixture of both tertiary binding -APP-IgGs and -A-L-E-V-P-T-DIgGs could increase the targeting potential of the vector. The purpose for using an AAV-2 as the Fig.1 (below): Exon 3: 5-CATGCCCTTCC-3 Exon 4: 5-GTACCCACTGA-3 Fig. 2 (right): Comparison of the wild type gene (green), and the mRNA transcript with protein sequence (red). The black line represents the exon for that region (left, 2; right, 4). Ref: NCBI: sequence viewer The resulting nucleotide sequence that spans the two exons, (21bp):

viral vector are for numerous reasons; this particular serotype already targets the CNS, there is a general lack of pathogenicity, and they can maintain long term trans-gene expression in animal models.4 Experimental Procedure The sequence identified in Fig. 2 will be used to construct an shRNA to be used in RNAi, Fig 3. As depicted in Fig. 4, the construct will include the Native APP promoter a space for the ribosome binding site, the targeted sequence, a loop sequence, the compliment sequence and a RNA Polymerase III termination sequence (poly A). This entire construct will be sent to an offsite lab to be constructed using nucleic acids, flanked on either side with the ITRs specific to the AAV-2 serotype. These ITRs are the only viral specific genes that needed to be added in cis4. This construct will then be inserted in trans with the AAV-2 helper genes with promoter, and the viral Rep and Cap genes, with a modification to the Cap genes, also Fig. 4. This modification includes adding genes for the Immunoglobulin-binding Z34C fragment of protein A, which will allow antibodies raised against the mutant protein and peptide sequence to bind to the outer surface of the viral capsid. This particular capsid protein binds the conserved, Fc, domain of the antibody; leaving the variable domain, antigen binding site, accessible

5-GCGCTGGAGGTACCCACTGAT -3 G/C content = 13/21 (61.9%) ~60%8

APP Isoform A APP Isoform H APP Isoform I APP Isoform D App Isoform G

MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHM 45 MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHM 45 MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHM 45 MDQLEDLLVLFINY-----VPTDGNAGLLAEPQIAMFCGRLNMHM 40 ------------------------------------MFCGRLNMHM 10

Fig. 5. This alignment of Isoforms A, H, I , D, and G illustrate that while some of the variants possess this sequence (A,H,I), others do not (D,G). The linked peptide sequence is in bold, ALEVPTD, and is the Target Sequence. Ref: ClustalW2 sequence alignment tool to interact with the mutant antigens. These antibodies will be raised in humanized mice by injecting them with a mixture of mutant protein and mutant peptide sequence. They will be harvested from the mice serum and purified using accepted ELISA techniques. The purified antibodies will be tested against several surface proteins of neuron cells, in order to prevent off site targeting in the brain. While this is not an issue of lethality, it is a goal of this project to ensure the lowest dose is needed to induce a healthy phenotype. Once the antibodies have been confirmed ineffective against other CNS surface proteins, they will be introduced to the rAAV-2 vector in vitro, under sterile conditions, and incubated for a period of 2 days. These recombinant AAV-2 vectors will only be recombinant for the IgG-binding protein and not the shRNA construct. The purpose of this is to determine the efficacy of cell targeting before actually inducing phenotypic expression. Allowing sufficient time for the antibodies to bind to all of the available IgG binding sites on the surface of the virus will help to ensure the highest level of cell-specific targeting. Once the incubation period is over, the rAAV-2 vectors will be transfected into mice that are transgenic for the AP protein, at various positions to determine targeting efficacy in vivo. These mice will need to be recombinant for the mutated APP isoforms, in order for the virus to target APP in the brain . 5 They will need to be given a rAAV-2 vector carrying the transgene for APP protein. Imaging of these tissues will be done, to confirm targeting, using Immunohistochemistry to locate viral particles using antibodies raised against them. Once it has been confirmed that there is an obvious correlation in favor of viral targeting of the mutant protein over healthy CNS cells possessing the wild type APP protein, the project will proceed forward with trials using the shRNA construct mentioned earlier in the project. These vectors will be used to transfect humanized mice directly into the blood stream, i.e. intravenously. If, however, there seems to be no appreciable increase in cell-specific targeting, then, in the interest of cost, normal AAV-2 vectors will be used to carry the transgene. These rAAV-2 vectors will be injected directly into the brain cavity of the mouse, to help an ensure CNS targeting by the viral vector. A control group of healthy mice will be reserved from the group used in the transfection, to compare against. Cognitive tests will be performed to determine if the treatment is successful in targeting the mutant proteins, and reducing the prevalence of AD. These tests will measure cognitive function of the APP recombinant mice treated with the rAAV-2 vector against mice that are APP recombinant without the treatment, and non-APP recombinant, but humanized, mice. Some mice from each group will be given current treatments for Alzheimers Disease and the efficacy of these treatments will be used as a baseline for comparison of the AAV-2 therapy. At the conclusion of 10 months, some of the mice that are recombinant for the APP protein will have developed advanced stages of Alzheimers Disease.6 At this point, mice from each group will be selected and euthanized, to test for amyloid plaque accumulation to determine if the treatment was effective at limiting their presence in the brain. The control for the effectiveness of the RNAi therapy will be a rescue experiment, where the researcher will reintroduce the mutant phenotype into mice that have been given the rAAV-2 vectored shRNA. The purpose of the control is to
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ensure that silencing the gene of interest causes the phenotype observed. The mice that have received the rAAV-2 will be inoculated again using a separate viral vector that is recombinant for mRNA that codes for the mutant phenotype. In order to avoid degradation by Dicer and RISC in these mice, this mRNA will not be an exact copy of the original mutant mRNA. The original peptide sequence will be maintained in the polypeptide chain but the actual codon sequence will be changed. i.e. codon(s) coding for the same amino acids will be used in place of the native codon(s); Fig. 6. This mRNA will be converted to linear single stranded cDNA and introduced into an identical mosaic AAV-2 viral vector as before, but now instead of being transgenic for the shRNA, they will contain the cDNA for the mutant phenotype. Administration of the therapy will be the same as the original rAAV-2 treatment for the shRNA. However, rather than using AAV-2 with altered tropisms, the wild type capsid proteins will be used, as they already target the CNS. At the conclusion of 10 months, if the mice that had originally not produced amyloid plaques are now forming them, then the original treatment must be considered the overwhelming cause of the observed phenotype. To confirm these findings, dissections of the mice brains will be performed and the level of plaque formation recorded. Also, learning and memory will be tracked over time to compare against the data collected in the original experiment. After which all of the data will be compiled and analyzed to fully determine the efficacy of both the RNAi and the vector targeting. Discussion of Significance This experiment has two, fairly distinct, objectives; both of which could provide useful information to the scientific community. The first is the RNAi of the known APP variants. Previous attempts at silencing dysfunctional forms of this protein have aimed at the amyloid region of the protein sequence. 7 This project intends to target the same variants but instead of targeting mutations in that region, the researcher intends to target sequences

upstream of that region. The reason being that studies have found that upstream mutations can augment the proliferation of amyloid plaques.9 By targeting mutations upstream of the dysfunctional subunit, the results may give insight into why these mutations are so detrimental. The second aim of this experiment is to develop a novel transducing vector for the expression of shRNA in target cells. By developing a mosaic rAAV-2 viral vector, utilizing immunoglobulin-binding domains to adhere to targeting antibodies, it may be possible to raise similar antibodies and improve targeting of similar viral vectors. By using current techniques for raising and purifying polyclonal antibodies, adeno-associated viral vectors of varying serotypes could be modified to target tissues, within their subset, more efficiently. Tissues such as cancers that form in various tissues that are already targeted by AAV serotypes could be targeted further. Helping to increase specificity opens up the opportunity for lower dosages, decreasing side effects and costs; often associated with these types of techniques. Therefore, should this technology prove useful it certainly could provide an avenue to researchers in the future to increase the specificity of targeting vectors like these, and increase the effect of potential gene therapies. While all of this remains speculation, there is certainly much to be gained by performing this experiment in its entirety. Fig. 6: Rescue amino acid seq. (mRNA to mRNA) mRNA: GCG\CUG\GAG\GUA\CCC\ACU\GAU AAS: A-L-E-V-P-T-D mRNA: GCA\CUU\GAA\GUU\CCA\ACG\GAC AAS: A-L-E-V-P-T-D
Converted to ssDNA: 5-GCA\CTT\GAA\GTT\CCA\ACG\GAC-3 Inserted in the shRNA sequence used in the rAAV-2 vectors for the rescue experiment. See Apendix A (Fig. 3 & 4) for more details of rAAVshRNA vector assembly. 4

References 1. Matsui. T, et al., Expression of APP Pathway mRNAs and Proteins in Alzheimers disease. Brain Res. vol. 1161, pp. 116-23 (August 3, 2007) 2. Zheng H, Koo EH, "The amyloid precursor protein: beyond amyloid". Mol Neurodegener. 1 (1) pp. 5 (July 3, 2006). 3. Stevenson, M., Therapeutic Potential of RNA Interference, The New England Journal of Medicine, vol. 351 (October 21, 2004), pp. 1772-1777 4. Gigout L, et al., Altering AAV tropism with mosaic viral capsids. Mol Ther. 11(6) pp. 856-65 (June, 2005). 5. Sturchler-Pierrat, Christine, Dorothee Abramowski, and Mairead Duke. "Two amyloid precursor protein transgenic mouse models with Alzheimerdisease-like pathology." PNAS94.24 (1997): 1328713292 6. Brummelkamp, T.R., Bernards, R., and R. Agami, Stable suppression of tumorigenicity by virus-mediated RNA interference, Cancer Cell, vol. 2, no. 3 (September 1, 2002), pp. 243-247 7. Rodrguez-Lebrn E, et al., Allele-specific RNAi mitigates phenotypic progression in a transgenic model of Alzheimer's disease. Mol Ther. 17(9) pp. 1563-73 (September, 2009). 8. T cell-specific siRNA Delivery Supresses HIV-1 Infection in Humanized Mice, Cell, vol. 134, no. 4 (August 22, 2008), pp. 577586 9. Kocherhans S, et al., Reduced Reelin expression accelerates amyloid-beta plaque formation and tau pathology in transgenic Alzheimer's disease mice. J Neurosci. vol. 30(27) pp. 9228-40 (July 7, 2010) 10. Miller VM, et al., Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles. Nucleic Acids Res. vol. 32(2) pp. 661-8 (January 30, 2004) 11. Crews L, et al., APP transgenic modeling of Alzheimer's disease: mechanisms of neurodegeneration and aberrant neurogenesis. Brain Struct Funct. vol. 214(2-3) pp.111-26 (March, 2010)

Appendix A: Supplemental Materials, Figures 3 & 4

Fig. 4 (Top): rAAV-2 vector assembly. The recombinant gene, the rep and modified cap genes, along with AAV-2 Helper genes are transfected into a host packaging cell (293 Human Kidney Cells). The cells are lysed, titers measured and purified. These vectors are then washed with antibodies to bind to the IgG-binding domains and finally injected into the mouse model. Fig. 3 (Bottom): The ssDNA sequence, the translated mRNA sequence being introduced into the cell, and the resulting shRNA formed by nucleic interactions between the palindrome sequences. This shRNA will be identified by DICER and RISC to inhibit protein expression of mRNA containing the Target Sequence.