Imaging Gene Expression in the Brain In Vivo in a Transgenic Mouse Model of Huntington's Disease with an Antisense Radiopharmaceutical and Drug-Targeting Technology

Imaging Gene Expression in the Brain In Vivo in a Transgenic Mouse Model of Huntington’s Disease with an Antisense Radiopharmaceutical and Drug-Targeting Technology

Hwa Jeong Lee, PhD¹, Ruben J. Boado, PhD¹, Dwaine A. Braasch, PhD², David R. Corey, PhD² and William M. Pardridge, MD¹

¹ Department of Medicine, UCLA School of Medicine, Los Angeles, California
² Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas

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FIGURE 1. (A) "Import-export" model showing bidirectional movement of 8D3 mAb to TfR. Bidirectional movement of PNA-8D3 conjugate across either BBB or BCM is possible because of ability of 8D3 mAb to access endogenous transport pathways for transferrin, which exist at both cellular barriers. Access to TfR pathways allows PNA radiopharmaceutical to move between blood and intracellular compartment of target cell. (B) Conjugation of PNA to 8D3 mAb to TfR creates bifunctional molecule that both accesses TfR for transport between tissue compartments and binds to target mRNA based on sequence specificity of nucleotide residues of PNA radiopharmaceutical. PNA has biotin moiety at amino terminus to allow for capture by conjugate of 8D3 mAb and SA and has carboxyl terminal tyrosine (Tyr) or lysine (Lys) residues to allow for radiolabeling with ¹²⁵I or ¹¹¹In, respectively.

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FIGURE 2. (A) Nucleotide sequence (in 5' to 3' orientation) of HD-PNA is bordered on amino terminus by biotin (bio) residue and by tyrosine (Y) and lysine (K) residues at carboxyl terminus. There are 5 linkers (designated O) flanking nucleotide sequence. Complementary nucleotide sequence of HD target mRNA (in 5' to 3' orientation) is shown, and methionine initiation codon (ATG) is underlined. HD exon-1 sequence is downstream of T3 RNA polymerase promoter (left solid box denoted by arrow), which allows for in vitro transcription of HD exon-1 mRNA. (B) Combined in vitro transcription/translation assays resulted in formation of ³H-labeled exon-1 huntingtin protein that was precipitated by TCA. Translation of HD exon-1 protein was inhibited in dose response by either PO-ODN (III) or by PNA. (C) RNase protection assay shows formation of HD mRNA protected fragment after complete nuclease digestion, because of hybridization of biotinylated HD PNA to huntingtin exon-1 mRNA (lane 2). Conjugation of antisense PNA to mAb-SA transport vector does not inhibit the hybridization of PNA to target mRNA, based on formation of RNase protected oligonucleotide shown in lane 4. Conversely, no protected fragment is observed after mixing of anti-luc PNA with HD RNA, either in unconjugated form (lane 3) or conjugated to mAb-SA vector (lane 5). BPB = bromophenol blue.

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FIGURE 3. BBB permeability-surface area (PS) product, AUC, and brain uptake, expressed as %ID/g brain, is shown for either unconjugated ¹²⁵I-HD-PNA or ¹²⁵I-HD-PNA-8D3 conjugate. Data are mean ± SE (n = 3 mice per group). Units of PS product are µL/min/g and units of 60-min plasma AUC are %ID·min/mL.

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FIGURE 4. Brain uptake, expressed as %ID/g brain, at 1, 2, 4, and 6 h after intravenous injection of ¹²⁵I-HD-PNA-8D3 conjugate is shown. Data are mean ± SE (n = 3 mice per group). Linear regression analysis yielded intercept (Int.) and slope values that are shown. PNA-8D3 conjugate underwent export from brain back to blood with t_1/2 of 4.3 ± 0.5 h.

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FIGURE 5. (A) Quantitative autoradiography (QAR) of 20-µm frozen sections of brain taken from 3 littermate control mice (top row) or 3 transgenic mice (bottom row) that were killed 6 h after single intravenous injection of ¹²⁵I-HD-PNA-8D3 conjugate. (B) Film autoradiography of 20-µm frozen sections of brain taken from 3 transgenic mice that were killed 6 h after single intravenous injection of ¹²⁵I-HD-PNA-8D3 conjugate (bottom row), and 3 transgenic mice that were killed 6 h after single intravenous injection of ¹²⁵I-luc-PNA-8D3 conjugate (top row). (C) QAR of 20-µm-thick ¹²⁵I-microscale standard strips is shown in inset. Integrated density for each standard is plotted versus known radioactivity for standard. (D) Integrated density obtained from scanning autoradiograms of brain sections taken from either littermate control mice or transgenic mice (A) was converted into measurements of organ radioactivity (mBq [µCi]/g) based on standard curve (C). Data indicate there is more than 3-fold increase in sequestration of brain radioactivity at 6 h after intravenous injection in transgenic mice compared with littermate control mice. Data are mean ± SE, n = 3 mice per group.