piggyLent: A Novel Hybrid Vector System for the Delivery of the CFTR gene Cameron Sweeney, Carlos Saavedra, Charles Wilkins

I. PROJECT SUMMARY:

Cystic Fibrosis (CF) is an autosomal recessive disease affecting one in 3,500 newborns. The disease is caused by a mutation of a single gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Of the six defined classes of mutations leading to dysfunctional CFTR function, class II (a deletion at F508 on chromosome 7) is the most common and leads to protein misfolding and thus a detrimental reduction in CFTR function. This reduction in CFTR function directly correlates to the degree of symptoms in the respective tissue. Homozygous individuals of the CFTR mutation exhibit a variety of debilitating symptoms primarily characterized by chronic inflammation and pulmonary infection. Because 90% of CF mortality is caused by pulmonary complications resulting from insufficient CFTR function, insertion of a functional version of the gene and prolonged, adequate expression has the potential to provide significant alleviation of life-threatening CF symptoms. Additionally, because heterozygotes for the mutation exhibit reduced CFTR function, yet are considered phenotypically normal, a goal of restoring CFTR expression to 50% of the non-mutant homozygous wild type would be sufficient to induce a normal phenotype. Furthermore, a mere 30% increase should lead to improvement in CF symptoms. Transfection of the CFTR gene and restoration of proper protein function, aside from providing amelioration of developed CF symptoms, offers great potential as a curative therapy for homozygous newborns prior to symptom onset.

Extensive research has validated viral vectors as effective methods of integrating genes of interest into host cells. One class of viruses that have been especially successful in terms of delivering DNA cargo are retrograde viruses, due to their inherent process of integrating their own DNA into their host genome. Lentiviral vectors such as HIV-1 and FIV have been shown to be better suited for gene therapy than other retroviral vectors due to their capacity to carry large cargo, be pseudotyped, integrate into nondividing cells, transfer genes with high efficiency and induce long term expression¹⁷. These viral vectors are also thought to be safer than other viruses due to the ability to remove the enhancer and promoter regions of their LTRs in order to prevent adverse changes in host genomic expression.

Cystic Fibrosis with its dysfunctional CFTR with a ΔF508 stems from a genetic modification arising from a deletion the phenylalanine. Because virtually all symptoms associated with the disease stem from the mutation of a single gene, there is great potential for amelioration of symptoms via therapeutic gene delivery systems such as viral vectors. Many studies have investigated the efficacy of adeno, adeno-associated, and retrograde viruses to integrate the CFTR gene into host cells in vitro, as well as in vitro for model animal^1–4.

II. INTRODUCTION:

The gene that causes Cystic Fibrosis was identified approximately 25 years ago. It is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. All 50 states are now screening newborn babies to detect the disease from early on. By detecting it early, therapeutic gene transfer can occur before the onset of inflammation and infection. CF affects several organs but primarily it is the lungs that are affected which are the cause of death in more than 90% of patients¹⁵. The lungs make a viable point of entry for any vector because it can be administered topically through the nasal passage; by using aerosolization, it will be spread to the pulmonary areas. The earliest clinical trials done on CF patients occurred in 1993 where viral and nonviral gene vectors were used in both the bronchial epithelial cells and the nasal passageway. In 2001, scientists and doctors from Imperial College London, Oxford and Edinburgh Universities, the Royal Brompton & Harefield NHS Foundation Trust developed a method of transporting a normal copy of the mutated gene to epithelial cells found in the lungs¹⁷. There were 136 patients total that did show better lung function. This was the first time that gene therapy was used to improve the function of the lungs.

This study seeks to utilize a PiggyBac (PB) transposon, which is a mobile genetic element that efficiently transposes vectors and chromosomes via a “cut and paste” mechanism. Transposable elements (TEs) are common among many organisms including: bacteria, plants, and humans. These elements have properties that enable them to persist the genomes of organisms. The powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRS in the PB vector to be easily mobilized into target genomes. Previous studies where researchers attempt to deliver a normal CFTR gene into the lungs have been successful, but the success rate is still very limited^16,17.

Transposons offer the ability to alter host genome in a more effective and precise manner. Previous studies have used Adenovirus and Adeno-associated virus in conjunction with piggybac transposons containing CFTR to cause recovery of cells ability to produce effective membrane potentials and restore protein function(piggy adeno). In addition, studies have harnessed the beneficial uptake of lentiviral vectors to deliver other types of transposons, such as sleeping beauty^5,6, for treatment of other genetic disorders.

Viral vectors have also been of high interest in the treatment of CF. In the forefront of these viral vectors, sits the Lentiviral vector which have been very promising in gene therapy because of their innate ability to infect both dividing and nondividing cells¹⁸. In addition, lentiviral vectors can integrate their genetic cargo directly into the chromosome of the target cell but not transfer sequences that encode for proteins derived from the packaging virus. This is key to preventing an immune response to the cells containing the transfer gene. Finally, lentivirus vectors can be pseudotyped to infect specific or a broad range of targets¹⁸. This study aims to utilize a lentiviral vector to deliver the therapeutic genes. They are very attractive vectors because of their large coding capacity, efficient gene transfer, persistent expression, ability to be easily taken up by many cell types, and lack of virus-encoded proteins that could cause adverse reactions from the immune system¹⁴. The main reason why it is being used for this study is because of the large coding capacity and the lack of virus encoded proteins.

Although traditional viral gene delivery methods are often successful, they do pose the possibility of harmful effects such as oncogenesis of host cells leading to cancer. HIV specifically targets integration sites which are actively transcribed, and this process can cause issues with regulatory proteins. While HIV causes replication issues, Adenovirus has a much higher necessary multiplicity of infection (MOI) for host tissue plasmid uptake. It has been suggested that this high MOI requirement is highly toxic. We aim to combine the usefulness and reduced MOI of the HIV particles uptake as well as use the successful piggybac transposon to cause a safer insertion of the CFTR. This study will provide initial proof of principal of a novel hybrid viral/nonviral gene delivery system, followed by evaluation of the vector’s utility in delivering the CFTR gene to human CF cells.

Unfortunately, one of the main limitations in this area of research is that there has not been many cases where gene therapy has been used due to the lack of viable FDA approved treatments. In addition, gene therapy elicits an inflammatory immune response that can be very strong or fatal. Clinical trials are also very expensive and difficult to perform.

III. RATIONALE AND SIGNIFICANCE:

Genetic disorders are a large area of recent research. With the human genome project, researchers and physicians are now able to identify what is specifically different about an individual at the DNA level. The next logical step for the health community is to treat diseases in new ways which relate the genetic information to the finished protein structure and how to address treatment and cures for individuals. This has been accomplished through treatments such as enzyme replacement therapy. These are innovative ways to alleviate the conditions associated with these diseases, however the best way to treat the patient would involve restoring the patient's DNA to its proper state of function.

Cystic Fibrosis stems from a genetic mutation, termed ΔF508, arising from a deletion of three nucleotides within the CFTR gene *. Because virtually all symptoms associated with the disease stem from the mutation of a single gene, there is great potential for amelioration of symptoms via therapeutic gene delivery systems such as viral vectors.

Many studies have investigated the efficacy adeno, adeno-associated, and retrograde viruses to integrate the CFTR gene into host cells in vitro, as well as in vitro for model animals. Based on the central dogma of molecular biology, inserting a functional gene into the host can result in alleviation of the most harmful conditions of CF.

The goal of the research is to restore CFTR gene using a treated lentiviral particle inserted with a transposon and transposase. The rationale is that the HIV particle with its high uptake ability and the piggybac transposase/transposon, which when inserted into random sites versus actively transcribing areas, reduces the likeliness of oncogenesis and increases the chance for a return of function.

Currently there is no treatment for CF. With the average life expectancy of 37 years, constant treatment of mucous buildup, infections, and organ failure, a cure is necessary to aid a huge population of people. With the insertion of functional genetic material this cure can be achieved. This lentiviral treatment will not only be a step forward for CF, it will also be a large step in genetic modification for diseases pertaining to genetic mutations. When put into similar practice treatments, this research could treat many genetic disorders and increase quality of life for many patients.

IV. RESEARCH OBJECTIVES AND HYPOTHESES:

This research aims to utilize lentivirus as a vector combined with a piggybac transposon and transposase for gene therapy in the treatment of Cystic fibrosis within stems cells that have a dysfunctional CFTR. Expectations are aimed at the use of lentivirus for it’s successful uptake within many cell types using a transposon with direct insertion into less translated regions of the genome reducing the chance of oncogenesis, and the usefulness for this method as a form of gene therapy.

If insertion of CFTR into the genome proves to be unsuccessful, the practice of combining both the piggybac and lentivirus is a method that should be tested because of its general significance as a genetic modifier. If this hybrid vector system does not perform well for CF, it may still aid in gene therapy for another disease with another protein whether it is located in the cell membrane, cytosol, or another organelle.

Therefore the Hypothesis Tested:

Human lung epithelial cells with ΔF508 mutation causing a dysfunctional CFTR protein, which causes a higher salt gradient extracellular space due to the lack of ionic movement, can be reconstituted via the transduction of a functional CFTR gene through the use of a hybrid lentiviral vector containing piggybac transposon/transposase. Gene insertion into the host genome will occur via translocation activity of the piggyBAC vector carried out by the action of expressed transposase iPB7, both delivered via a lentiviral vehicle. Successful translocation will be evident by reappearance of functional CFTR within the membrane and the lowering the salt concentration in the extracellular space.

V. METHODS:

VI.

Cell Culturing conditions

• HEK293T cells will be cultured as previously described in Dulbecco’s modified Eagle’s medium, 10% fetal calf serum, penicillin, and streptomycin^5,6.
• Primary epithelial cultures of CF cells will be grown through isolation of human CF epithelial tracheal cells via enzymatic digestion as previously described7,8.

Production of piggyLent System Vectors

Integrase-Deficient Lentivirus (IDLV) components:

• The IDLV backbone will consist of the mutated packaging plasmid pCMVΔ8.74 D64V6,9, envelope plasmid encoding vesicular stomatitis virus glycoprotein pMD.G2, and empty lentiviral plasmid (Addgene).

Transposon piggyLent vector (pL-CFTR) production:

• The piggyBac plasmid, pPB-mCherry/Puro-CMV-hCFTR, will be designed in silico and constructed by VectorBuilder.
• The cassette to be transposed will be inserted between the terminal repeats of pPB and include the functional CFTR gene. mCherry and puromycin resistance genes will be included as selection markers.
• The cassette will then be cloned from pPB into the IDLV backbone between the EcoRI and XbaI restriction sites6. (Fig. 1)

iPB7 Transposase (helper) vector production:

• To produce a separate hybrid vector to deliver transposase activity, hyperactive insect piggyBac transposase (iPB7) will be cloned into the IDLV vector from the pcDNA3.1/myc-HisA plasmid 6,10 (ThermoFisher) at the EcoRI and Xhol restriction sites. (Fig. 1)
• iPB7 will be driven by the CMV promoter of the IDLV, and not incorporated into a piggyBac cassette.

HEK293T transfection:

• HEK293 cells will be co-transfected with separate transposase (iPB7) and transposon (piggyLent) vectors to induce transposition of CFTR, mCherry, and puromycin resistance genes. (Fig. 2).
• Varied concentrations of each vector will be administered and allow for analysis of dose-dependent transfection success.

Transposition validation

• To validate transposase directed integration of the transposon cassette, transfected HEK293T cells will be assessed via flow cytometry for expression of the mCherry gene 48 hours following transfection.
• Additionally, to determine the quantity of viral product that can be harvested from transfected cells, Gag p4 titer will be quantified using Retro-Tek HIV-1 p24 Antigen ELISA kit (ZeptoMetrix, Buffalo, NY).

Optimization of vector concentrations

• HEK293T cells will be selected for puromycin resistance through a colony formation assay.
• Greater number Puromycin resistant colonies will represent greater transposition of the translocon
• Assay will be assessed based on varying dilutions of both piggyLent and transposase vector to determine the optimized ratio of transposon:transposase to be delivered.

Treatment of CF cells

Production and harvest of piggyLent for CFTR integration

• The CFTR gene will contain a silent mutation in order to differentiate b/t the integrated and endogenous versions of the gene via RT-PCR following CF cell transduction.
• HEK293T cells will be co-transfected as described above with the determined optimal vector ratios. · 48 hours later, successfully integrated cells will be selected for through colony formation in puromycin. · Puromycin resistant colonies will then be ultracentrifuged, and the supernatant will be harvested and filtered through a .45 µm filter5.

Transduction of CF cells and analysis of CFTR function

• Cells will be transduced with the harvested vectors via baso-lateral application as previously described7.
• Successfully transduced cells will be selected via colony formation in puromycin as described above.
• To assess post integration transgenic CFTR expression levels, silent mutations will have been inserted into the vector’s CFTR cDNA in order to distinguish the transgenic and host mRNA through RT-PCR
• cAMP-stimulated chloride currents will then measured using Ussing chambers12 at 2 week intervals for 16 weeks and compared to non-transduced cells.
• Based on previous studies^7,13, an increase in chloride current to levels of ~ 4 µA/cm2 will likely be considered significantly different from non-transduced cells and assessed as restored CFTR function.

plasmid fig.png

Fig 1. Schematic of vector production. To create the piggyLent vector for transposon delivery, piggyBac plasmid (Vectorbuilder) containing CFTR and selection marker cassettes will be cloned into an IDLV (Addgene). To allow for transposase directed host-integration of the cassettes, iPB7 from pcDNA3.1/myc-HisA plasmid (ThermoFisher) will be cloned into an IDLV to produce a helper vector to express transposase. Screen Shot 2015-11-22 at 2.45.35 PM.png

Fig 2. Schematic of piggyLent mechanism. Cotransduction of a cell with piggyLent-CFTR and it’s iPB7 transposase allows integration of CFTR gene into host genome. Transposition is mediated by the expression of the transposase, which acts to integrate the gene cassette (CFTR) into the host genome to provide long term expression. Figure credit to Vink et al6.

VI. RESEARCH TIMELINE: See Attached VII. BUDGET:

Source of Funding: Cal Poly, San Luis Obispo CSU School System Source of Supply Year: 2016-2017 Line Item Description

Amount Micropipettes X5 Quasar Instruments $ 495.00 Petri Dishes 500/Case GreenBio $ 86.00 .45 µm membrane filters Sigma-Aldrich $ 174.90 HEK293 (TCells) X5 Clontech $ 325.00 Dulbecco’s Modified eagles medium Sigma-Aldrich $ 282.00 Flow cytometer Boston Industries $ 875.00 PiggyBac Vector Plasmid X5 Vector Builder $ 195.00

pCMVΔ8.74 D64V Addgene $ 65.00 Envelope plasmid pMD.G2 Addgene $ 65.00
Empty Lentiviral Plasmid Addgene $ 65.00 Retro-Tek HIV-1 p24 AntigenKit X5 Biolabs INC $ 2950.00 PCDNA3.1/myc-HisA plasmid X2 Thermofisher $ 1280.00 RT PCR X3 Thermofisher $ 624.00

Total $ 14,593.00

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9. Burnight ER, Staber JM, Korsakov P, et al. A Hyperactive Transposase Promotes Persistent Gene Transfer of a piggyBac DNA Transposon. Mol Ther Nucleic Acids. 2012;1:e50. doi:10.1038/mtna.2012.12.

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