Assessing an Adult Zebrafish Model for in Vivo Delivery of Morpholino Oligomers

Assessing an Adult Zebrafish Model for In Vivo Delivery of Morpholino Oligomers

by Joseph Yoojong Kim

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Biochemistry and Biophysics (Honors Scholar)

Presented May 17, 2018 Commencement June 2018

AN ABSTRACT OF THE THESIS OF

Joseph Yoojong Kim for the degree of Honors Baccalaureate of Science in Biochemistry and Biophysics presented on May 17, 2018. Title: Assessing an Adult Zebrafish Model for In Vivo Delivery of Morpholino Oligomers.

Abstract approved:______Hong Moulton

Antisense morpholino oligomers are synthetic macromolecules can modify gene expression. While these compounds have great potential to treat a broad range of human diseases, they suffer from poor cellular delivery. Conjugating cell-penetrating peptides onto these morpholinos is one option of addressing delivery, however development remains slow due to the cost of the animal models used to assess in vivo delivery and safety of peptide-conjugated morpholinos. We have produced a novel transgenic zebrafish model that express gene transcripts for blue fluorescent protein.

If the morpholino can successfully enter the nuclei, transcripts for red fluorescent proteins will also be expressed. Through retro-orbital injection, it was found that a particular cell-penetrating peptide was able to deliver a morpholino to zebrafish heart by a transcription-detecting assay. The results demonstrate that this zebrafish model has potential to be used an alternative vertebrate model to identify effective yet safe peptide-conjugated morpholinos for future pharmaceutical applications.

Key Words: Morpholino, PMO, peptide-conjugated morpholino, PPMO, zebrafish, gene transcription, exon-skipping, BFP, RFP.

Corresponding e-mail address: [email protected]

Assessing an Adult Zebrafish Model for In Vivo Delivery of Morpholino Oligomers

by Joseph Yoojong Kim

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Biochemistry and Biophysics (Honors Scholar)

Presented May 17, 2018 Commencement June 2018

Honors Baccalaureate of Science in Biochemistry and Biophysics project of Joseph Yoojong Kim presented on May 17, 2018.

APPROVED:

______Hong Moulton, Mentor, representing Biomedical Sciences

______Robert Tanguay, Committee Member, representing Environmental & Molecular Toxicology

______Indira Rajagopal, Committee Member, representing Biochemistry and Biophysics

______Toni Doolen, Dean, Oregon State University Honors College

I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request.

______Joseph Yoojong Kim, Author

Table of Contents

SECTION I: Background ...... 1

SECTION II: Materials and Methods ...... 7

Materials ...... 7 Retro-Orbital Injection ...... 8 Fish Dissection and RNA Extraction ...... 9 Measuring BFP and RFP Transcripts ...... 10

SECTION III: Results and Discussion ...... 12

Visualization of Injected Zebrafish ...... 12 Transcription of BFP Gene...... 14 Transcription of RFP Gene ...... 17

SECTION IV: Conclusions and Future Directions ...... 19

SECTION V: References ...... 21

SECTION I: Background

Phosphorodiamidate morpholino oligomers (PMOs or morpholinos for short) [1,2] are synthetic macromolecules that are capable of modifying gene expression. PMOs do this by inhibiting mRNA translation, modifying pre-mRNA splicing or interfering with miRNA maturation and activity of a targeted gene. The structure of PMOs is similar to that of a canonical nucleic acid oligomer, with a few key differences that promotes its stability and uncharged structure (Figure 1). Nucleic acids contain five- carbon sugar rings (ribose or deoxyribose) that carry nitrogenous bases (adenine, cytosine, guanine, thymine or uracil), linked together with phosphodiester bonds.

PMOs however utilize six-carbon morpholine rings that carry the bases, coupled together through phosphorodiamidate groups. The phosphorodiamidate linker carries two phosphorous-nitrogen bonds: one is between the phosphorous and the nitrogen of an adjacent morpholine ring and another is between the phosphorous and a dimethylamine group. This linker provides charge neutrality through its non-ionic bonds, which stand in contrast to the anionic charged backbone of canonical nucleic acids [1]. PMOs are typically designed to be 18-31 nucleotides in length with each nucleotide bearing an A, T, G, or C base; this gives an average molecular weight of a

PMO to be approximately 8000 Daltons, depending on the specific sequence it is designated for. PMOs have been found to be stable in basic conditions and sensitive in acidic conditions at pH < 3. PMOs have also been found to be resistant to nuclease degradation [3] and cannot be degraded in human serum, cells [4], and tissue lysates

[5].

DNA PMO

Base = A, G, C or T

Figure 1. A side-by-side comparison between a DNA molecule and a PMO molecule.

PMOs function by binding to their target RNA through complementary anti-parallel base pairing (Figure 2) in the cytosol and/or nucleus of a cell. Such binding prevents other molecules from interacting with the RNA and therefore interfering with certain bioprocesses from taking place [1-2, 6]. While PMOs have the overall effect of altering gene expression, the specific mechanism by which such a process occurs is dependent on the specificity of the target sequence. Such specific mechanisms can include blocking mRNA translation, modifying pre-mRNA splicing, blocking miRNA maturation and activity, or blocking other RNA sites including splice regulation sequences, ribozymes, translocation signals, and slippery sequences [7-13].

Figure 2 illustrates the PMO’s most common mechanisms of action: blocking mRNA translation or modifying pre-mRNA splicing. Because of both the mechanism and stability of the compound, PMOs carry promising therapeutic potential, as they can be used to treat a broad range of human diseases, including viral, bacterial, age-related and genetic diseases. One example is Eteplirsen, a PMO-based drug that was

approved by the FDA in late 2016 to treat the degenerative muscle disease Duchenne

Muscular Dystrophy.

Figure 2. PMO’s most common mechanisms of action PMOs, initiated by complementary binding of the compound to mRNA.

However, therapeutic applications of PMOs are limited by poor cellular uptake.

PMOs demonstrate poor abilities to cross the plasma membranes of cells because of their molecular weight of about 8500 Daltons and possessing no known transporter that can shuttle PMOs inside of cells. One way to remedy this is to conjugate a cell- penetrating peptide (CPP) onto either end (5’ or 3’) of the PMO. The easiest way to conjugate the CPP onto the PMO is through an amide linkage, although it is possible to utilize disulfide, thioester, or ether linkages [14-15]. The composition of these

CPPs continues to be a matter of research; however, CPPs that have already been studied can be made up of various amino acids and derivatives such as arginine,

phenylalanine, and 6-aminohexanoic acid. These CPP-PMO conjugates, also known as peptide-conjugated PMO (PPMOs), have significantly increased cellular uptake compared to PMOs. PPMOs can bind to cell-surface proteoglycans, which are then subsequently taken up by endocytosis [15].

While PPMOs have been successfully tested in rodents to enter the bloodstream and knock down selected genes, there is a need for an alternative animal model to study various compositions and structures of CPPs on the resultant activity and safety of

PPMOs [16]. This is because housing and testing rodents is time-consuming, expensive, and wasteful due to the large PPMO doses required. Zebrafish (Danio rerio) can be considered a viable alternative because smaller doses can be used to assess for PPMO activity. This will make studies more economical, and the resulting affordability of experimental designs using larger treatment groups will give more statistically significant data. This is shown by how the zebrafish, which shares 70% of the human genome, has been used as a model organism for various diseases and toxicology [17-20]. Historically, injections of compounds into zebrafish were done intraperitoneally; however, such a technique can carry a high mortality rate because it can pierce major organs or put the fish at risk for infection at the injection site [21-

22]. Therefore, there is a need to have an alternative injection method to administer

PPMO to the blood stream of fish for accuracy and minimum mortality. Here, we have developed a detail method of retro-orbital injection for administration of PPMO.

The method was adapted from a published method [22] with modifications.

To assess for PPMO activity in zebrafish, a transgenic zebrafish (PMO-Switch Fish) was generated that contains a dual reporter system that can switch fluorescence signal from blue color to red color through PPMO treatment. This PMO-switch fish, developed in collaboration with Dr. Karl Clark at Mayo Clinic, produces a transcript containing an in-frame BFP cassette with a stop codon in one exon and an RFP cassette in the next exon. The BFP-RFP transcript is expressed ubiquitously from a beta-actin promoter. When produced together in one mature mRNA the BFP is expressed but RFP will not be translated. When a splice-modifying PMO binds the transcript, the BFP cassette exon with its stop codon is spliced out, permitting RFP to be translated (Figure 3).

Figure 3. The PMO-Switch zebrafish that was developed to test for the efficacy of PPMOs. Blue fluorescent protein would normally be produced, however if the PMO is successfully delivered red fluorescent protein (RFP) will be expressed instead due to successful exon-skipping by the PMO on the (BFP) transcript.

Therefore, both downregulation of blue florescent protein (BFP) and upregulation of red fluorescent protein (RFP) can be monitored in the fish if the exon-skipping PMO

is successfully delivered to the tissue of interest. The model is designed to be easily visualized, requiring only a simple fluorescent microscope to observe changes in gene expression (Figure 4) for transparent zebrafish embryos and adult fish. For non- transparent adult fish, the PMO-switch effect can be assessed through gel electrophoresis and sequencing and quantitatively measured through techniques such as quantitative polymerase chain reaction (qPCR).

Figure 4. Comparisons between PMO-injected and un-injected zebrafish embryos, visualized through fluorescent microscopy. The top photo displays visualization of BFP, where the treated-embryo exhibits less fluorescence because of BFP knockdown. The bottom photo displays RFP expression, which is up-regulated in the PMO-treated embryo.

We hypothesize that the adult PMO-Switch zebrafish can serve as a cheap and efficient in vivo model to determine the efficacy, biodistribution and toxicity of

CPPs for delivery of PMOs. Our long-term goal is to identify tissue-specific

peptides that are suitable for delivering PMOs to tissues of interests for various disease indications. From the zebrafish model, we hope to find that PPMOs are capable of gene expression changes inside the animal. We also expect that there may be indications of organ-specific targetability from the PPMO based on the specific structure of the CPP. The objectives of this project are to 1) develop a method to administer PPMO through the retro-orbital route, 2) develop a method to assess BFP and RFP transcripts in organs, and 3) determine efficacy and tissue distribution of a PPMO. This project will help serve the long-term goal of developing PMO-based pharmaceuticals to treat infectious and/or neuromuscular diseases.

SECTION II: Materials and Methods

Materials

The PMO-Switch zebrafish were housed and maintained at the Sinnhuber Aquatic

Research Laboratory (SARL) in Corvallis, Oregon. Tricaine solution was prepared and provided by Carrie Barton at SARL by mixing 400 mg tricaine with 97.9 ml distilled water and 2.1 mL 1M Tris, adjusted pH to 7.5 with HCl. This solution was further diluted to safely anesthetize the fish by mixing 4 mL of stock solution with

100 mL of fish water. PMO (5-ATTCCGGCTGAACTGTAAATGAATG-3’) was purchased from Gene Tools, LLC and its peptide-conjugate was made by Dr. Hong

Moulton’s lab. Sterile 1X phosphate-buffered saline (PBS) was used to solubilize and dilute PPMOs. Dextran Texas Red (70 KDa), cDNA reverse transcriptase kits,

RNase inhibitor, SYBR® Select Master Mixes, qPCR plates and adhesive film were

purchased from ThermoFisher Scientific. For procedures calling for water, UltraPure

DNase/RNase-free water was used, purchased from Invitrogen. RNAzol, used to extract RNA from sample tissue, was purchased from Molecular Research Center,

Inc. RNA purification columns, tubes, and buffers were purchased from Zymo

Research. 4-Bromoanisole (BAN) was purchased from Sigma Aldrich. qPCR primer sets for Beta-actin, BFP, and RFP were purchased from Integrated DNA

Technologies. qPCR was performed on a StepOnePlus™ Real-Time PCR System.

FlashGel™ cassettes, dock, extraction buffer, marker, and ladder were purchased from Lonza. Sequencing services were ordered from the Center for Genome

Research and Computing at Oregon State University.

Retro-Orbital Injection

Each zebrafish was weighed by transferring to a tared beaker filled halfway with fish water. The zebrafish were then grouped in equal populations based on similar masses to ensure that the injected dose of PPMO is consistent for all zebrafish. Based on the average weights for each group, a 2x concentration of the PPMO injection solution was calculated to deliver 4 µL of a 25 mg/kg dose to each group. These 2x PPMO concentrations were made by diluting lyophilized PPMO with 1X PBS. The injection solution was made by mixing one volume of 2x PPMO (50 mg/kg) with one volume of 25 µM Dextran Texas Red. A 10 µL Hamilton® 700 series syringe was washed with 70% ethanol and PBS prior to withdrawing 4 µL of injection solution. Zebrafish were anesthetized by placing them in Tricaine until tail fin ceased movement and breathing was slowed. Placing the anesthetized fish on a foam pad, the needle was

positioned with the bevel facing up such that if the fish’s eye was a clock, the needle is positioned at the 9 o’clock position at a 45 degree angle (Figure 5). The needle was inserted behind the eyeball 1-2 mm deep and the contents of the syringe were slowly injected. After 5 minutes to allow the PPMO to distribute throughout the body, the zebrafish was then brought to a fluorescent microscope to determine if the red fluorescence from the Texas Red dye can be observed in the blood stream of the fish as a method of validation. This procedure was then repeated until all zebrafish were properly injected, where the fish were then housed and monitored to see if adverse reactions occurred from the procedure.

Figure 5: Material can be injected behind the zebrafish’s eye at a 45 degree around the 7-9 o’clock position relative to the eyeball.

Fish Dissection and RNA Extraction

After a 48 hour housing period, the zebrafish were euthanized through an ice bath submersion containing 5 parts ice to 1 part water on a fish net for 10 minutes until opercular movements ceased, following NIH requirements. The zebrafish were then dissected and extracted for brain, eyes, gills, heart, liver, muscle, and tail, which were all each placed in their own respective Eppendorf safe-lock tube and stored with dry ice. These organs were then mixed with 500 µL RNAzol and homogenized with 0.5

mm zirconium oxide beads in a Bullet Blender® Homogenizer for three minutes at speed setting 8. The homogenized organs were then mixed with 200 µL UltraPure

DNase/RNase-free water and centrifuged at 12,000 rcf for 15 minutes to recover the

RNA-containing supernatant. From the supernatant, 550 µL of the RNA-containing supernatant was subsequently mixed with 2.5 µL of BAN and centrifuged at 12,000 rcf for ten minutes to remove any other traces of genomic DNA. A volume of 100% ethanol was then mixed with an equal volume of supernatant and transferred to a

Zymo RNA purification column to be washed and eluted following the manufacturer’s protocol. The concentration and purity of RNA was then assessed on a NanoDrop™ spectrophotometer, where an A260/A280 ratio of ~2.0 was indicative of a highly pure RNA sample.

Measuring BFP and RFP Transcripts

After diluting the RNA to 200 ng/µL with DNase/RNase-free water, all seven organ samples were reverse transcribed to cDNA following the protocol for ThermoFisher’s

High Capacity cDNA Reverse Transcription Kit in an Applied Biosystems Veriti

Thermal Cycler. These cDNA samples were then diluted four-fold to prepare for qPCR. Each cDNA sample was tested in triplicates for gene transcription of BFP,

RFP, β-actin (for positive control), and a negative control. For each set of genes,

SYBR® Master Mix buffer was mixed with forward and reverse primers of the respective transcription following the manufacturer’s protocol, multiplied by the number of samples that can fit into a MicroAmp EnduraPlate Optical 96-well Fast

Clear Reaction Plate (Table 1). To this reaction plate, 16 µL of a single

transcription’s SYBR reaction mixture was dispensed into each assigned well.

Afterwards, 4 µL of cDNA was dispensed to each appropriately assigned well. After sealing and centrifuging the reaction plate, the plate was placed in an Applied

Biosystems StepOnePlus Real-Time PCR machine. The qPCR program was run with a holding stage set at 95°C for 10 minutes and a 45-cycle stage that was set at 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds to obtain a melt curve, 60°C for 1 minute, and 95°C for 15 seconds. This qPCR program was run for approximately three hours using 6-carboxyfluorescein (FAM) as the reporter dye and a Nonfluorescent-Minor Groove Binding quencher (NFQ-MGB).

Gene Expressed Forward Primer Reverse Primer β-actin 5’-AAG CAG GAG TAC 5’-TGG AGT CCT CAG ATG GAT GAG TC-3’ CAT TG-3’ BFP 5’-CAG CTA GTG CGG 5’-CAT GGT TAC CTT ACG AAT ATC ATC TGC C-3’ CTT CTT CTT TGG-3’ RFP 5’-CAG CTA GTG CGG 5’-CCT TGA AGC GCA TGA AAT ATC ATC TGC C-3’ ACT CCT TGA T-3’ Table 1: The primer sequence for the three genes whose transcription were measured by qPCR for PPMO-injected zebrafish organs

After qPCR was completed, the reaction plate was stored while the data was collected and analyzed for CT values. Absolute expression was used to determine RFP transcription as opposed to relative expression because the RFP gene will only be expressed under the condition of successful PPMO-induced skipping of the BFP exon. To further assess the validity of possible RFP transcription, gel electrophoresis was performed for the RFP-targeted qPCR products of all seven organs. On Lonza’s

FlashGel™ 8+1 Well Double Tier 2.2% Recovery Cassette, 4 µL of qPCR product was combined with 1 µL of loading dye and pipetted into the wells along with a

FlashGel™ 100-4000 bp DNA marker ladder. Following the manufacturer’s protocol, the FlashGel™ cassette was inserted into The FlashGel™ dock system and run at 150

V for ten minutes. After the gel was imaged and analyzed on a UVP GelDoc-It®2

310 Imager, the bands that appeared on the gel were extracted with FlashGel™

Recovery Buffer and sent over to Center for Genome Research and Biocomputing for sequencing. The sequence results were then compared to the known RFP sequences that were implemented inside the zebrafish to see if the PPMO-induced splice modification has occurred.

SECTION III: Results and Discussion

Visualization of Injected Zebrafish

After retro-orbital injection, the zebrafish was observed under a fluorescent microscope to see if the Dextran Texas Red dye that was mixed with the PPMO was found to be distributed throughout the entire body. With an absorbance wavelength of

595 nm and an emission wavelength of 615 nm, the fluorescence of the dye can be easily observed with the correct filter. If fluorescence was found to be distributed throughout the entire body, this would mean that the PPMO has been successfully delivered through retro-orbital injection. Indications of a successful injection were made by observing fluorescence throughout the entire zebrafish body, extending from the gills to the tail (Figure 6).

Figure 6: Fluorescence that is extended throughout the entire body, especially in the vascular system, is indicative of a successful retro-orbital injection.

Comparisons between the fluorescence of the injected zebrafish and previous studies of transgenic fluorescent fish shows that the injected zebrafish has a fluorescent compound that was successfully delivered to the vascular system [23]. Using the fluorescent tracer eliminates the variable on whether the compound was delivered successfully and allows accurate interpretation of a change in BFP/RFP gene transcription attributed by the ability of a cell-penetrating peptide to deliver PMO.

Red fluorescent signal continued to appear for a week after the injection upon injecting PPMOs mixed with Dextran Texas Red. However, this level of strong circulation cannot be guaranteed for the PPMO, as the two compounds were mixed with each other for injection, not conjugated. This means that there is a chance that the PPMO may not be entirely distributed throughout the zebrafish, owing to the different properties between the compound and the dye it was mixed with. However, visualization of red fluorescence can at least confirm that the PPMO-dye mixture was successfully delivered (at least initially) into the blood stream of zebrafish through retro-orbital injection.

Transcription of BFP Gene

The RNA that was extracted from zebrafish brain, eyes, gills, heart, liver, muscle, and tail were reverse-transcribed to cDNA and analyzed by qPCR to determine gene transcription of BFP and RFP. As the BFP exon was engineered to be the primary transcript expressed in the transgenic zebrafish, observing BFP transcription for all seven organs will confirm the validity of the BFP-RFP zebrafish model. Transcription is measured by the threshold cycle number (CT), the number of cycles that it takes for the sample fluorescence signal to be above the background signal. From each organ,

CT values were collected for BFP and β-Actin for the groups that were treated with

PPMO and the untreated group (Figure 7).

A B

Figure 7: The CT values for the BFP gene and the housekeeping gene β-Actin for all seven organs between the (A) untreated group and the (B) treated group shows that while β-Actin expressions are mostly consistent, BFP expressions are not.

The CT number for BFP were normalized to ΔCT against β-Actin, a universal protein that is highly conserved across all vertebrate [24]. Quantifying BFP transcription ΔCT numbers for all seven organs will establish a baseline that can be used to determine relative quantification of BFP (Figure 8).

Figure 8: The transcriptions of the BFP gene on all seven organs for untreated fish and PPMO-treated fish, represented by ΔCT through β-actin normalization, demonstrates the validity of the zebrafish BFP-RFP model.

The availability of ΔCT of BFP for all seven organs indicates the success of the BFP-

RFP zebrafish model, as it highlights the transcription of the BFP gene. β-Actin transcription, which is used as a baseline for transcript comparisons, was found to have CT values between 18-22 across all organs, with the tail producing the most β-

Actin transcripts and the liver producing the least. Interestingly, ΔCT measurements are shown to be different across the entire spectrum of organs, ranging from small to large differences between BFP and β-Actin transcription. Higher ΔCT numbers are indicative of having fewer BFP transcripts, as there is a larger transcription difference between the housekeeping gene and the target gene. For this particular experiment, the reasons behind these diverse ΔCT numbers among the organs are currently unknown; however, it can be proposed that the transcription of BFP depends on the

entire transcriptome of the organ cell type itself. In fact, the differences in the ΔCT profile for BFP transcripts between untreated zebrafish and PPMO-treated zebrafish were also found to be organ specific (Table 2). When ΔΔCT was calculated as

ΔCT(PPMO-treated) – ΔCT(Untreated), it was found that ΔΔCT values for brain, gills, heart, liver, and muscle were negative while ΔΔCT values for eyes and tail were positive. Because ΔΔCT was calculated with respect to PPMO-treated ΔCT, positive

ΔΔCT values are indicative of gene knockdown, while negative ΔΔCT values imply an increase in gene transcription. However, such differential transcriptions could also be due to the contamination of non BFP-carriers in each zebrafish group. Therefore, it is currently difficult to use BPF transcript level to determine PPMO efficacy, although this can be alleviated with better genotyping studies. Future studies that investigate the entire transcriptome of each organ on the BFP-RFP zebrafish, such as RNA-Seq, will give further insights in the transcription profile unique to the organ and the changes within it that occurs after the PPMO is injected.

Organ ΔΔCT = ΔCT(PPMO) - ΔCT(No PPMO) Brain -1.30 ± 0.8 Eyes 0.78 ± 1.3 Gills -3.04 ± 0.9 Heart -3.53 ± 0.8 Liver -2.57 ± 0.6 Muscle -1.75 ± 0.7 Tail 0.60 ± 0.6 Table 2: ΔΔCT for BFP between treated and untreated zebrafish shows differential transcription patterns that are unique to each organ.

Transcription of RFP Gene

The success of the CPP design can be measured by assessing the transcription of the

RFP gene. RFP will only be expressed if the PMO has successfully entered the plasma membrane of the cell for its exon-skipping activity, a task that is only possible through the success of the CPP to deliver PMO into nuclei of cells. The CPP can also be assessed for its organ specificity, which can have future applications in studying infectious and genetic diseases that are localized to a specific location in the body.

RFP transcription was analyzed using absolute quantification because of the gene’s rarity that will only appear under certain circumstances, which is the success of

PPMO activity (Figure 9).

A. Amplification Plot 10 1 Heart 0.1 Muscle

0.01 Rn

Δ 0.001 0.0001 0.00001 NC 0.000001 2 6 10 14 18 22 26 30 34 38 42 Cycle

Figure 9: (A) qPCR amplification data shows that the PPMO-treated heart exhibits RFP transcription at a CT number of 36. Out of all of the organs tested, the heart was the only organ that showed viable detection of the RFP gene. (B) Melt curve data of the zebrafish heart confirms that the detection of the RFP gene is sound.

Out of the seven organs that were tested, only the zebrafish heart was found to express RFP. Such transcripts were detected at a moderate level, with a CT number of

36 and a ΔCT value of 14 set against β-Actin (data not shown). Melt curve analysis of

this transcription gave a single sharp peak, which indicates that there is only a single, pure amplicon. To further ensure that the amplified gene in qPCR is RFP, the qPCR product of the heart was extracted and run on a gel along with the other six organs that were expressed for RFP (Figure 10).

Figure 10: Gel electrophoresis of the RFP-expressed qPCR product of PPMO- injected zebrafish shows that only the heart showed transcription.

Gel analysis of the PCR products show only a single band that is approximately 100 bp in length from the zebrafish heart, which is within a similar range of the RFP primer set products that were designed by Dr. Clark. To truly confirm that this is the

RFP gene, the band was extracted and sent to the CGRB for sequencing with its respective primers. These sequences were then aligned with the expected primer product sets on BLAST to check whether the alignments correspond to the RFP gene that were engineered into the zebrafish model (Table 3).

Total Score Query Cover E-Value Ident Forward Primer 145 100% 1E-40 100% Reverse Primer 137 100% 2E-38 100% Table 3: BLAST alignment between the PCR product sequence and the expected sequence shows that the sequences for both primers maintain a good query cover while having very low E-values.

BLAST alignment shows that the extracted sequence from zebrafish heart and the expected sequence align with good query cover and a low E-value, which represents the number of matches that it takes to achieve the same score by chance. This goes to confirm that the extracted gel band was indeed the amplified RFP gene.

This series of experiments validates that upon injecting this particular PPMO, the zebrafish heart will express the RFP transcript. It remains unknown as to why only the zebrafish heart was able to successfully express RFP upon PPMO treatment, due to the novelty of the experimental model. One possibility may be because the zebrafish heart simply has the most access to the hemodynamic forces that carries the

PPMO throughout the bloodstream, which increases the likelihood of successful entry and activity [25]. It may also be that the properties of the particular CPP that was conjugated onto the PMO conferred properties that allowed organ-specific targets.

Nevertheless, the transcription of RFP demonstrates proof of concept that the BFP-

RFP zebrafish serves as an efficient in vivo model to study the efficacy of PPMOs, which would have normally not been possible due to the splicing mechanism behind the engineered fish.

SECTION IV: Conclusions and Future Directions

The results of this study shows that the BFP-RFP zebrafish can be used as a new model that can be used to study the effects of PPMOs in vivo. This was validated from the success of PPMO delivery through retro-orbital injection and the confirmed transcriptions of BFP and RFP at an organ-specific basis. However, the causes behind

the organ differentiation in BFP transcription and the organ-specificity of RFP transcription remain unknown. Studies on the transcriptome, such as DNA microarrays or RNA-Seq, for each organs can help determine transcription profiles to give insights as to how differential transcriptions of BFP come about. Testing different conjugates of PPMO can help determine the features behind CPP design that can be used to test for different types of organs.

Based on the establishment of this model, further studies can be conducted that provide further information on the properties of PPMOs and their effects on gene expression in vivo. Conducting dose-dependent studies on efficacy and toxicity will help establish therapeutic indexes on a wide selection of PPMOs. In addition, other techniques can be used to better understand the mechanism and kinetics behind

PPMO activity itself, such as fluorescent microscopy or cell cytometry for subcellular distributions. These studies, made easier with the new transgenic zebrafish model, will help towards the long-term goal of establishing pharmaceutical applications of

PPMOs for treatments in genetics disorders and infectious diseases.

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