The ABCA3 gene encodes a lipid transporter protein in type ll alveolar cells and is essential for proper surfactant production and lamellar body formation. Mutations cause Congenital Surfactant Deficiency leading to impaired surfactant production, respiratory failure in neonates and interstitial lung disease.

Gene therapy is an emerging treatment for a wide range of genetic illnesses and shows great promise for treatment of ABCA3- related disease. It would allow for correction of gene mutations, providing a long term solution.

Cells that are treated with gene therapy to repair ABCA3 will produce substantially more surfactant than untreated, ABCA3 mutated cells.

Congenital Surfactant Deficiency  Overview

• Congenital surfactant deficiency is an umbrella term for a group of genetic diseases characterized by insufficient production or dysfunction of pulmonary surfactant. 
• While it’s rare it’s associated with severe respiratory distress in full term infants, and early onset lung disease. 

ABCA3 Mutations

• These mutations account for >50% of genetic surfactant dysfunctions, making it the most common form. 
• Present in 1 in 20,000-40,000  live births, 1 in 33-70 are carriers. 

• Over 200 known ABCA3 mutations. These can disrupt surfactant production by:

• preventing the protein from being made 

• causing the proteins to misfold 
• damaging its ability to transport lipids

Symptoms and Clinical Manifestations

• Severe work of breathing
• Cyanosis
• Cold limbs and digits 
• Tachypnea
• Grunting
• Low oxygen levels 
• Tachycardia
• Mottling

In rare cases, CSD may not be identified until late infancy, childhood or even adulthood. While ABCA3 babies are typically critical from birth other forms can present as idiopathic chILD or adult pulmonary fibrosis. 

Treatments  There is no cure, current treatments focus on sustaining life until a lung transplant becomes available. This is rare.

• Oxygen therapy 
• Surfactan replacement therapy
• Nutritional support 
• Inhaled corticosteroids/ systemic steroids
• Macrolide antibiotics 
• Non invasive ventilation (CPAP, BiPAP)
• Mechanical ventilation (conventional, high frequency oscillation, jet)
• ECMO

Most babies don't make it to one year of life. Only 10% of transplant candidates live long enough to receive new lungs. Of those, only 30% make it >10 years. Oftentimes a second transplant is needed due to infection, rejection and surfactant degradation. 

ABCA3 Gene Overview

• The ABCA3 gene is located on chromosome 16 at 16p.13.3 (petite arm, region is 13.3). 
• It’s a negative strand (transcribed from complementary DNA strand).
• It’s 33.6 kilobases (kb), mRNA length is 6.8kb. 

Mutations (over 200 known)

• Missense (single amino acid changes).
• Nonsense (early stop to protein creation).
• Frameshift (insertion or deletion of nucleotides, everything downstream is changed).
• Splice (a change in the DNA sequence that messes with the signals that tell the cell where to cut and join RNA to make mRNA).

• Insertions/deletions (general term for insertion/deletion of nucleotides, doesn’t matter if they’re frameshift). 

• Mutations in A3 are associated with multiple conditions including neonatal distress syndrome, childhood interstitial lung disease and adult pulmonary fibrosis. 

• Mutations can be identified with genetic testing, lung biopsy or electron microscopy. 
• It’s a monogenic inheritance (single gene), predictable patterns of recessivity, meaning two pathogenic mutations, meaning two pathogenic mutations are needed. 
• Carriers may be asymptomatic or have mild lung issues. 

Shulenin et a;. (2004) examined lung tissue from ABCA3 mutated patients and observed abnormal lamellar bodies, suggesting ABCA3 is important for lamellar body formation. 

The ABCA3 Protein  Overview

• The gene encodes a 1,704 amino acid polypeptide. It’s an ATP-binding cassette, subfamily A member 3 (typically refers to proteins that deal with lipid transport. 
• Phospholipid transporter located on the membrane of lamellar bodies . 
• Once the protein is made it’s sent to the endoplasmic reticulum where it's folded into the correct shape. It then goes to the golgi apparatus to undergo glycosylation (addition of sugar molecule) this is necessary for the protein to work). Finally it’s cut at the N-terminal to remove an extra piece. Beforehand the protein weighs 190kD, afterwards, 150kD. 
• Covers about 10-20% of lamellar body membranes. 
• Has a two transmembrane domain and a two nucleotide-binding domain. 

Transport

• ABCA3 acts as a bridge between the cytoplasm and lamellar body. 
• Uses ATP to change its shape, then translocates (flips) lipids across the membrane. 
• This is because the amphipathic structure of lipids makes it difficult for them to cross the hydrophobic core of the lamellar membrane.
• Because A3 helps shape the lamellar body, mutations can cause them to be small or absent. 

Surfactant Composure Pulmonary surfactant is a mixture of 80% phospholipids, 10% neutral lipids and 10% surfactant proteins. 

• SP-A: regulates surfactant secretion.
• SP-B: spreads surfactant.
• SP-C: stabilizes lipid layer.
• SP-D: binds to pathogens. 

Secretion

• It’s excreted by exocytosis, the lamellar body fuses with the plasma membrane of type ll alveolar cells. Surfactant is released into the lining fluid. 
• Once secreted contents undergo structural changes. Lamellar body contents unfold into a lattice structure called tubular myelin.
• It spreads into a single layer at the air-liquid interface. This is the surface active agent.

Other

• Up to 90% is recycled but it does degrade over time. 
• It’s taken up into the type ll cells via endocytosis and cleared by alveolar macrophages. 

Type ll Alveolar Cells Overview 

• Also known as type ll pneumocytes or alveolar epithelial type ll cells. 
• 17% of the alveolar wall, 5% of alveolar surface. 
• Each cell has a surface area of 250μm2
• Alveolar fluid is (roughly) 35mL per adult lung, PH is 6.9. 

Purpose

• Responsible for production, storage and secretion of pulmonary surfactant.
• Cube shaped, smaller than type l but more numerous.
• Help trigger cytokines and antimicrobial peptides (for protection against pathogens). 
• Can separate into type l after injury to help lungs repair and regenerate. 

Creation

• Appearing around 24 weeks of gestation, surfactant production increases dramatically during the third trimester. 
• Labour increases surfactant production. 
• Women at risk for preterm labour may be given corticosteroids, these help mature type ll cells, therefore; surfactant production.  

Lamellar Bodies Overview

• Secretory organelles found (mainly) in type ll alveolar cells.
• Typically 0.2-1.1 micrometers, oval shape (sometimes irregular).
• Not visible under light microscopy unless stained, transmission electron microscopy is considered the gold standard for visualization. 
• Membrane bound, filled with layers of surfactant proteins and phospholipids and surrounded by a limiting membrane filled with transport proteins. 
• Breathing, stretching and adrenaline trigger lamellar bodies to excrete surfactant. 
• Lamellar bodies are transported through the cytoplasm to the type ll cell. 

Formation

• Begins in the golgi apparatus where lipids and proteins are directed into multivesicular bodies, these will become lamellar bodies. 
• ABCA3 transport protein moves phospholipids into the developing lamellar body. 

CRISPR-Cas9 Gene Therapy What is CRISPR-Cas9

• Clustered regularly interspaced short palindromic repeats (CRISPR).
• Gene editing tool that allows scientists to modify the DNA of living organisms. 
• Originally discovered in naturally occurring bacteria and was adapted for laboratory use. 

How does CRISPR-Cas9 work

• Made up of two parts, a small strand of RNA programmed to look for a specific DNA sequence, and Cas9, an enzyme that cuts a double strand of DNA. 
• Recognition: once CRISPR has entered the cell it works to locate the DNA it’s been told to find. It uses a PAM sequence (a short sequence required for Cas9 to identify target) once found, crRNA binds to the complementary DNA sequence. 
• Cleavage: Cas9 cuts both DNA strands 3 base pairs up from the PAM, creating a double stranded break. 
• Repair: Once DNA is broken the cell will work to repair it. A donor DNA template can be introduced into the cell. This new template aligns with the cut DNA and the cell copies the correct sequence into the genome. 

CRISPR Delivery Methods

• Electroporation; uses electric impulses to create openings in the phospholipid bilayer of the cell membrane. 

• Seen in CASGEVY

• No risk of viral integration 
• Can lead to cell death, risks are especially high for neonates.

• Suitable for ex-vivo

• Adeno-associated virus 

• Popular for CRISPR in-vivo

• Low risk of immune response 

• Too small to carry ABCA3 

• Lentiviral vectors 

• Big enough to carry ABCA3

• Can be modified to be able to easily infect more cells.
• Integrates into cells= longer lasting, but cancer risk. 

• High risk of immune response, though typically mild.

• Adenoviral vectors

• Large carrying capacity 

• Work in dividing and non-dividing cells 
• Can infect a wide range of cells
• High risk of immune response 
• Not integrating, might not be great long-term.

Controlled

• Confirmed ABCA3- related surfactant deficiency.
• Databases from which information was collected (all approved).

Independant

• Type of treatment given: gene therapy vs current therapies.

Dependant

• Level/quality of surfactant produced.

The procedure for this project was carried out in three sections; background research, data analysis and application.

Section 1: Background Research A comprehensive review of peer approved, scientific literature collected from reliable databases was carried out. This allowed for a thorough understanding of all project components including: congenital surfactant deficiency, the ABCA3 gene and protein, CRISPR-Cas9 based treatments and various gene therapy delivery methods. This is crucial for proper execution of my project.  Section 2: Data Analysis  All data was synthesized and analyzed to help determine the feasibility of gene therapy as a treatment for ABCA3- related disease. This stage also allowed for assessment of potential challenges/risks.  Section 3: Application  In this step I assessed ABCA3 gene therapy’s prospect for clinical application. Using the information gathered I proposed a possible treatment utilizing CRISPR gene therapy for congenital surfactant deficiency caused by mutations in the ABCA3 gene.  Materials

• Zotero: used for collection and creation of citations.
• Microsoft excel: information was entered in to help with data analysis and to help visualize information.
• Publicly available images were used (and cited) for visuals.
• Scientific databases (NIH, Pubmed, NHS…)
• Canva

I completed an in-depth review of publicly available literature collected from peer reviewed studies on approved scientific databases. The literature reviewed consists primarily of papers on ABCA3 related disease, existing and emerging treatments for congenital surfactant deficiency, and gene correction strategies with further research into ex-vivo compared with in-vivo methods.

ABCA3- related congenital surfactant deficiency is the common form affecting 1-20,000-40,000 live births. Over 200 known variants with the majority being missense or private (unique to families or individuals). Babies born with biallelic homozygous ABCA3 mutations die shortly after birth, those with compound heterozygous mutations may develop interstitial lung disease later on. Survival >5 years of age without a transplant is rare, >20 is almost unheard of. Patients present not only with changes to the protein and its ability to transport lipids, but also abnormal lamellar bodies, suggesting ABCA3 plays a major role in proper lamellar development. Typical presentation is severe neonatal respiratory distress in a full term infant that has little to no improvement with endotracheal surfactant. Survival without a lung transplant is extremely unlikely, and even with one the five year survival rate is only 60%.Treatment options are limited and temporary, focused on preserving life rather than curing the disease. Given the implications of ABCA3 mutations, novel treatment options are needed.

Gene therapy provides a novel treatment for a wide variety of inherited diseases. It has been approved as a treatment for conditions such as sickle cell anemia, acute lymphoblastic leukemia and more. Multiple delivery strategies are available making it a suitable option for a wide range of patients. Can be done ex-vivo (outside the body) or in-vivo. Treatments with gene therapy can be curative. 

Despite promise, genetic therapy comes with considerable risks and challenges. Therapies using viral vectors may use integration which can cause cancer, or they may trigger an immune response. Some non viral delivery methods can lead to cell breakdown or death, these risks may be elevated in neonatal populations. Delivery itself may pose a challenge as the airway has natural barriers in place though pre-treatments can help mitigate this. Additionally, a clear, detailed explanation to guardians is necessary as there may be objections or concerns on their part regarding safety of gene therapy especially with viral vector use. 

Congenital surfactant deficiency caused by mutations in the ABCA3 gene leads to defective phospholipid transportation in type ll alveolar cells. Treatment options are limited and survival is rare- even with a lung transplant. A definitive treatment is needed to increase survival and decrease the burden on health systems. ABCA3 is expressed in alveolar type ll cells by transporting phospholipids into lamellar bodies as well as playing a role in lamellar body formation, making it critical for proper surfactant production. >50% ABCA3 function may greatly improve survival rates and quality of life. Some mutations (missense, nonsense) may be more treatable than others because they only involve a single nucleotide. Gene therapy provides a possible curative treatment which would allow for survival into adulthood with limited need for further medical intervention. Unlike a lung transplant degration of surfactant would not be a concern. Vector selection plays a substantial role in gene therapy success with carrying capacity being the biggest consideration. Lentiviral vectors are promising due to a combination of large vector capacity and DNA integration. It has shown high success rates in-vivo in lung epithelial cells with pre-treatments to decrease barriers, however they do risk an immune response though usually mild. Adenoviruses may be too small or too high risk. Non viral vectors are a possibility but often carry a risk of cell death which is more significant in neonatal populations. Another important consideration is in-vivo vs ex-vivo. Ex-vivo allows for screening before reintroduction to improve safety and allows for precise editing however extraction of tissue can be difficult, especially from lung cells. In-vivo is preffered as it's less invasive and reduces the amount of required specialized equipment. It’s also superior in neonates due to their underdeveloped immune systems. Its versatile and long acting, however there are risks of off target effects as control is limited. Preclinical research suggests gene therapy is a viable treatment option, however research is limited. Neonates with severe mutations die shortly after birth, children and adults who survive have interstitial lung disease and a greatly reduced life span. This places considerable stress on not only patients but families and healthcare systems. Surfactant disorders are also a leading cause of lung transplants in infants. While it carries risks, benefits greatly outweigh them. Before gene therapy can be applied in a clinical setting more research is needed to assess efficiency in different mutations and to develop eligibility criteria; however it shows significant promise as a future treatment.

Congenital surfactant deficiency that causes fatal neonatal respiratory distress syndrome in full-term infants and interstitial lung disease. Treatments are limited to temporary, life preserving measures or lung transplant. Future direction should focus on choosing a suitable vector and applying gene therapy in a clinical setting. Furthermore, success should lead to development on gene therapy for other forms of congenital surfactant deficiency. This will allow for long-term survival of these populations and increased quality of life, as well as decreased burden on caregivers and healthcare systems. 

ABCA3- related congenital surfactant deficiency is a fatal genetic condition with very few available treatments. With up to 1 in 33 individuals being carriers, a definitive cure is desperately needed.

I have designed a possible CRISPR based gene therapy for treatment of CSD. When compared to current available therapeutic strategies it performs better in virtually every category. My strategy utilizes lentiviral vectors as the delivery method due to their superior size and use in-vivo. Since neonates have underdeveloped immune systems, immunogenicity is low, and in-vivo alteration reduces stress placed on the body. Another consideration is time, since ABCA3 mutated patients experience early mortality, timely administration of treatment is imperative, in-vivo treatments are considerably shorter than their ex-vivo counterparts. Below is an outline to how my treatment strategy works:

1. Lentivirus RNA is edited to contain guide RNA, Cas9 enzyme, a PAM sequence and a donor template. Any viral causing genes are removed.
2. The lungs will be pretreated with mucolytics to improve accessibility.
3. The lentivirus will then be introduced through an endotracheal tube where it will bind to cells.
4. After fusing with the cell membrane, it will release its RNA into the cytoplasm.
5. Inside it will be converted into double stranded DNA and enter the nucleus, integrating into the genome.
6. The gRNA binds to Cas9 and locates the DNA sequence adjacent to PAM and binds.
7. Cas9 then creates a double stranded break in the DNA.
8. The cell now works to repair the cleave. It can use the healthy donor DNA template to do so, though this will not be 100% effective as it must compete with the cells' faster, natural repair technique.
9. This should be a one time treatment

• Publication bias: small number of studies and incentive to publish good results may overestimate effectiveness and underestimate risks. 
• Small sample sizes: ABCA3- related disease is rare and there is a limited number of individuals in studies. 
• Animals vs humans: many gene therapy studies are done using animal models which have differing characteristics to humans. 
• Limited long term data: gene therapy in use for ABCA3 is new and very limited, this is an issue because long-term efficiency and side effects are unknown.

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I would like to thank all of the individuals who helped contribute to this project. First I would like to express my gratitude to my family who supported and encouraged me throughout this entire process. To the authors, whose publications made this project possible, and to Dr. Howlett, who let me shadow her, the experience inspired me in my project selection. Thankyou to everyone, you are the reason for this projects success.