�� Berkeley Pharma Tech Journal of Medicine Correspondence:� KBLPCPD��!HNBJM�DPN Keywords: $01% (FOF�UIFSBQZ (FOFUJDT 1VMNPOBSZ�EJTFBTF -VOHT� %FMJWFSJFT� /BOPQBSUJDMFT Submitted�.BZ��, 202���� "ccepted�JVOF��� ������ 1VCMJTIFE�+VMZ��� ����� Full Open Access Creative Commons Attribution� License 4.0 Abstract Gene therapy is a growing field in research and development that may offer a long-lasting solution to several complex diseases, including chronic obstructive pulmonary disease (COPD). COPD is characterized by chronic inflammation in the lungs and the airways, leading to respiratory problems. COPD includes chronic bronchitis and emphysema. Optimizing treatments for gene therapy in COPD is of paramount importance given COPD's prominence as the fourth leading cause of disease-related death in the United States. We reviewed delivery methods in the current research, including liposomes, nanoparticles, electroporation, adeno-viruses, and adeno-associated viruses (AAV). The broad customizability in the diagnostic and treatment methods is evident in the recent studies. In this paper we explore each method and/or biomarker and evaluate several gene therapy avenues for COPD. (FOF�5IFSBQZ�Avenues for $ISPOJD� 0CTUSVDUJWF�1VMNPOBSZ�%JTFBTF �ïw�£×ÙåØ��×âÛäÚßäÛu�¬ÛéÞ×��×èê×u�£ëÚÛ��ÞÛèÜßâéu�¨ã×è�¬×ßÚ and ¯×äâå×ä�§ÝëïÛä Background Chronic obstructive pulmonary disease, also known as COPD, is characterized as a chronic in�ammatory lung disease that causes obstructed air�ow from the lungs. It includes multiple progressive lung diseases that a�ect millions of people yearly. In 2018, COPD was the fourth leading disease related cause of death in the United States, 6.4% of Americans were diagnosed with the disease, unfortunately, this number is likely higher due to undiagnosed cases1. Figure 1: Diagram of the lungs and the effect chronic bronchitis and emphysema has on them. Boxed under chronic bronchitis are images of healthy (A) and unhealthy (B) bronchial tubes, (B) shows the inflammation and increased mucus in the unhealthy bronchial tubes. Boxed under emphysema are images of the alveoli, healthy (A) and unhealthy (B). In the unhealthy (B) image there is a membrane breakdown due to inhaled pollutants that causes larger sacs with a decreased surface area. COPD is not caused by a single gene or factor, this makes a single treatment for all patients less e�ective, and makes an appeal towards a more personalized treatment1. Two of its major contributors include emphysema and chronic bronchitis. Chronic bronchitis is characterized by the in�ammation of the bronchial tube lining and excess mucus production, the bronchial tubes are responsible for carrying air to and from the air sacs (alveoli) within the lungs. Emphysema is a condition in which the alveoli membranes are destroyed due to chronically inhaled pollutants. The membrane breakdown creates larger air sacs rather than smaller ones, Berkeley Pharma Tech Journal of Medicine | 59 this decreases the surface area and the amount of oxygen that can di�use into the blood. There are many factors that cause the development and progression of COPD, the main factors include cigarette smoke and work-related pollutants2. COPD is also considered to be inheritable, as there are genetic components that can play a role in disease development. The �rst gene identi�ed to be associated with COPD was SEROINA, which encodes alpha1-antitrypsin (A1AT). De�ciencies in A1AT can lead to emphysema, but only 1-3% of COPD patients have the de�ciency. Additionally, it has been recently discovered that altered miRNA expression in the lungs might also play a role in the COPD mechanism. The characteristic symptoms that de�ne COPD include the following: breathing di�culty, coughing, wheezing, oxidant/antioxidant imbalance, emphysema (alveolar wall destruction), mucus hypersecretion, enhanced cytokines, chemokines, protease, and in�ammation3. Current conventional therapeutic strategies of COPD utilize antioxidant and anti-in�ammatory drugs3. These drugs tend to be bronchodilators, such as β-agonists and muscarinic antagonists, and inhaled corticosteroids, both of which are only used for short-term management. These treatments only target the patient’s symptoms, and do not stop or reverse damage to the lungs2. Bronchodilators, such as β-agonists and muscarinic antagonists work to relax the muscles in the lungs to alleviate coughing and make breathing easier, whereas inhaled steroids reduce airway in�ammation and help prevent exacerbations. Working towards a treatment for COPD that could prevent or reverse lung damage, would impact the lives of millions. Gene therapy is an attractive alternative to current treatments, it is able to deliver medications and therapeutics to speci�c target sites within the lungs. However, it's important to note that there are limitations due to various biological barriers. These limitations can include; o�-target vector e�ects, genetic material, and delivery e�cacy1. Due to the variety of infections causing diseases in the respiratory system, there are only 17 FDA approved treatments available for patients, none of which treat lung diseases1. ABECMA is an example of an approved gene Berkeley Pharma Tech Journal of Medicine | 60 therapy that treats multiple myeloma, a disease where the body’s white blood cells proliferate and cause harmful build ups throughout the body. This speci�c gene therapy modi�es the patient’s T-cells to attack the cancerous white blood cells4. Another example includes Luxturna, a viral vector gene therapy form that treats congenital blindness. Most patients with congenital blindness contain a mutation in the RPE65 gene, which produces the chromophore 11-cis retinal protein that is vital for eye function. Luxturna works by delivering functional copies of the RPE65 gene to the retinal epithelial cells5. Cystic Fibrosis (CF) is a common candidate for gene therapy research and can o�er some insight in the treatment of COPD as well. Due to defective sodium/potassium ion channels in the ciliated cells, the airway becomes dehydrated and starts secreting more mucus which is a breeding ground for infection-causing bacteria. Because of the fatality of the airway obstruction, doctors are researching to replace the CFTR gene during the neonatal period to maximize the success of the therapy and the patients’ quality of life6. To deliver the CFTR gene into the lung, scientists have experimented with the oral inhalation of aerosolized vectors using a nebulizer rather than a liquid �lled nasal tube. This method of delivery reduces the risk of aspiration and could be useful when treating other lung diseases, including COPD6. Modulator therapies are also burgeoning methods to treat CF, and each therapy targets a speci�c mutation in the CFTR gene. The success of the modulator therapy was shown in a research study when 55% of the subjects experienced signi�cant decrease in P.aeruginosa7. Genes that contribute to COPD such as SERPINA1 that causes an α-1 antitrypsin de�ciency would be a good candidate gene for potential modulator therapies with certain mutations in the gene8. Recent genetic, biochemical and histological evidence also suggests altered transforming growth factor beta (TGF-β) signaling is associated with COPD development and progression. TGF-β regulates the respiratory system and can lead to diseases if it is mutated. Subduing altered TGF-β signaling in the airways and alveolar sacs via gene silencing technologies may provide similar Berkeley Pharma Tech Journal of Medicine | 61 therapeutic outcomes to modulator therapies and other treatment options9. Deliveries The development in gene therapy for COPD hinges on several factors, one of the most prominent of which is delivery method to ensure e�cacy and precision in use of the particular therapy. Delivery methods must optimize performance and transfection as well as safety both for the patient and protection of genetic material. Even in common inhalable drug delivery, limitations are present due to the complex defense mechanisms of the respiratory system. These factors are limited in e�cacy and primarily focus on airway obstruction via anticholinergics and dual β2-dopamine 2 receptor antagonists10. Techniques used for gene therapy can be separated into 3 major categories: chemical, physical, and viral. Chemical techniques entail the non-viral methods for creating materials and particles used to transfect or insert genetic material into the target cells. This can be achieved in several ways whether it be to weaken the cell function or to create new vectors that take advantage of common cellular functions and mechanisms, such as liposomes and nanoparticles (NPs). Next, physical techniques revolve around mechanical methods to typically achieve new access points or alter the function of the cell slightly without introducing a chemical alteration or manipulation to the equation. Increasing the permeability and uptake of genetic material of the cell or direct injection is the most common mechanism in which these methods are able to insert the genetic material. The more prominent of these is a shock treatment to open the cell membrane for a brief period of time known as electroporation (EP). Thirdly, viral delivery methods are created by modifying viral genomes to take advantage of viral mechanisms of inserting genetic material. The most common vectors that have extended into the realm of gene therapy for pulmonary diseases are EP, liposomes, viral vectors, and NPs. Each of the aforementioned methods give way to their own advantages and problems; seen in Figure 2, thus validating the need for further research on gene therapy for symptoms of COPD. [JG1] Longevity and �nding the ideal carrier to inhibit the e�ects of COPD using these treatments is necessary to Berkeley Pharma Tech Journal of Medicine | 62 counteract the lifelong e�ects traditionally associated with it and other commonplace pulmonary diseases. Figure 2: Modeled from diagram of delivery method choice for gene therapy in the lungs [1]. The more generally observed shortcomings and advantages of each key delivery method to be discussed in detail. Individual modifications, especially in the case of liposomes and NPs, can be made to better these such as particle coating and more effective targeting mechanisms. Non-Viral Methods Liposomes Taking advantage of natural lipid bilayers to employ liposomes with genetic material, lipoplexes, for delivery cargo to cells utilizing endogenous functions of the cell membrane is one of the primary strategies in gene therapy for COPD. The most glaring functional advantage of lipofection as a technique is the lack of immune response and cytotoxicity upon transfection that has been observed11,12. In addition to this, low cost and ease of use make liposome based gene therapy a preferred treatment. Morphology which mimics that of cell membranes’ lipid bilayer can be modi�ed with targeting molecules to adhere to di�erent cells with more precision and limited collateral damage. The accepted but not completely understood mechanism of action takes advantage of random Brownian motion of the liposomes upon entrance into the cell to e�ectively disperse the material13. In generic drug delivery this allows for seamless in�ltration and dosing of the cell. However, the speci�city of gene delivery Berkeley Pharma Tech Journal of Medicine | 63 requires nuclear transport of the contained genetic material; these shortcomings can be observed through endocytosis inhibitors and endosome trackers to visualize delivery sites14. The endogenous nature of liposomes is massively important for pulmonary diseases and speci�cally COPD where constant in�ammation and exacerbations take place regularly. A well observed phenomena since the 1970s, liposomal immunogenicity, can be built upon even further to trigger minimal immune responses. For example, liposomes for drug delivery that showed an inverse correlation with immunoglobulin (IgM) in the brain were modi�ed to enhance absorption of IgM and thus the immune response and overall e�ectiveness of the treatment was improved15. Relative to the lungs, clinical trials have utilized lipoplexes in tandem with a plasmid carrying human CFTR in inhalable doses to limit mucus problems in patients with CF, a common comorbidity of COPD16. The bene�ts of these native characteristics of liposomes are signi�cant but can present their own pitfalls as well which must be monitored. Natural mechanisms can act as impediments to treatments such as the bovine pulmonary surfactant Alveofact have been shown to weaken Berkeley Pharma Tech Journal of Medicine | 64 Figure 3: Image shows the mechanism by which liposomes (and some NPs) can introduce genetic information into a cell by taking advantage of endocytosis, a natural cell mechanism, and limiting the immune response– thanks to the endogenous nature of the liposomal structure and makeup. several types of lipoplexes17. Overcoming these innate obstacles while maintaining the key bene�ts of lipoplex usage is of paramount importance and needs more research to fully understand the functionality in di�erent cell types within the pulmonary system. Speci�c work with cells and the mechanisms of the pulmonary system will yield a better understanding of how lipofection can be used as a widespread treatment. Nanoparticles NPs (nanoparticles) are a massive and growing �eld of research for a number of possibilities including gene therapy for the lungs. Nanoparticles for gene therapy can be characterized by submicron sized particles that Berkeley Pharma Tech Journal of Medicine | 65 optimize surface to volume ratios while maintaining good performance of biocompatibility and biodegradation3. The customizability of NPs for di�erent cell types, degrees of accumulation, and degradation o�er several advantages as carriers for gene therapy treatment in the pulmonary system. Taking advantage of endocytosis and permeability factors of cells in similar manner as lipoplex-based techniques, NPs can utilize similar lipid based qualities but are ultimately de�ned by size and their diverse makeup which extends beyond liposomes. Targeting cells and material within the airways limits the abilities to penetrate into the tissue for many delivery methods. As mentioned, liposomes can encounter problems with penetrating surfactant and mucosal layers due to degradation and dissociation. Modi�cations can be made to NPs to resist mucoadhesion and trapping or degradation that may otherwise occur. One of the most common methods for this is a coating of polyethylene glycol (PEG) polymer to provide a hydrophilic and neutral coating to counteract traditional charge based dynamics used in synthesis18. It has been found that these PEG coatings provide varying levels of improvement in penetration of the thick mucus that exacerbates the issue of access to airway epithelial cells in patients with advanced COPD; degraded structures such as neutrophils and dense meshes of highly negative charged structures create a less permeable mucus layer19,20. These �ndings further the ability to overcome airway mucus, one of the primary issues in inhalable gene therapy to treat COPD and prevent signi�cant worsening of symptoms. PLGA A candidate in gene therapy using NPs is that of poly ((d,l-lactide-co-glycolide) (PLGA)) based nanoparticles. PLGA-NPs are FDA and European Medicine Agency approved for drug delivery systems, not gene delivery. Good results have been demonstrated regarding biocompatibility that exceeds that of common liposome based treatments in transfection of HepG2 cells for gene delivery21,22. Surface modi�cations can be made to PLGA-NPs to enhance e�ciency in performance such as the addition of polyethyleneimine (PEI) made by Bivas-Benita et al. to enhance performance in the pulmonary system given its merits as a gene delivery Berkeley Pharma Tech Journal of Medicine | 66 vector; the particles were seen to escape the endolysosomal envelope and subsequent damage to continue release of material in some instances as well23. Dendrimers A relatively unique NP structure that has gained exposure in gene therapy treatment given success in diagnostics is dendrimers. These NPs are based on the dendrimer polymers that exhibit a radially symmetric pattern of branching chains. This allows for many exposed ends at the surface that can be modi�ed much like other NPs to optimize their function as a delivery method for genetic material. Hypotheses revolve around the potential for these large amounts of ionizable branches to be modi�ed, most commonly utilizing poly-(amidoamine) and poly-(propyleneimine) as a base, or immediately paired with genetic material for testing29. To help in structure stability and formation the aforementioned PEG coating is applied to dendrimer NPs prior to material attachment1. Given the plentiful terminals, rapid absorption and dissipation of the NPs is another problem solved via these PEG coatings by enhancing retention by the lung tissue. The majority of the published work on dendrimer NPs in lung tissue gene therapy presents them in the context of this ‘PEGylation” given the ability to control uptake with a much higher degree of precision than without30,31. Dendrimers share in the success of NPs as a whole in their continued research and have signi�cant potential for delivery of gene therapy to inhibit COPD symptoms provided the state of knowledge continues to advance. Electroporation Electroporation (EP) di�ers from previously discussed delivery methods in its physical based mechanism of action rather than the chemical basis on which NPs and lipoplex therapies rely. It has emerged as one of the few physical methods researched for gene delivery given its safety and e�cacy compared to others. EP takes advantage of an electric �eld applied across cells to alter permeability and allow previously injected genetic material to be more readily endocytosed. Transfection e�ciency in tumors using EP has provided the foundation of knowledge for its usage in lung tissues. Intense Berkeley Pharma Tech Journal of Medicine | 67 tissue analysis and/or electric �eld testing is required in most cases to ensure optimal settings given the natural variation in biological tissues32. Traditional chemical based approaches make up the vast majority of research due to customizability. However, as these �elds move forward experiments have validated EP as a comparably e�ective method when physiological responses are considered in tandem with pure gene expression amongst cells33. Viral Methods Viral vectors have been researched as avenues for gene therapy for years now given their development in the early 1970s and 80s and ability to take advantage of mechanisms native to the viruses themselves. The primary vector types found in modern gene therapy for COPD and lung tissue oriented studies are adenovirus and adeno-associated virus (AAV) based approaches. The di�erences in viral vectors and e�cacy is contingent on the exact protein capsid and tissue tropism, the gene of interest for therapeutic purposes, and the alterations that control the gene expression for the vector34. These work together and can be chosen or altered to perfect one’s vector for its desired purpose. Common problems brought about in viral vector usage despite modi�cations is the innate immune response. T-cell responses and toxicity within tissue is a cause of viruses that has developed to help the body respond. However, redesign and manipulation of these viral genomes and bodies can still carry markers to inhibit their expression when used as vehicles for gene therapy. Adenoviruses With regard to the aforementioned characteristics adenoviruses are a family of icosahedral nucleocapsid viruses. They are unenveloped and hence carry their genetic material, double stranded DNA, within this characteristic icosahedral chamber. Adenoviruses account for several acute respiratory illnesses and human adenoviruses (HAdVs) have a wide range of categorized species based on key characteristics that number over 80 types35. Work in gene therapy for COPD stems from HAdVs’ versatile tissue tropism and prevalence in lung-related diseases and replication36. Imaging techniques Berkeley Pharma Tech Journal of Medicine | 68 have revealed signi�cantly higher carrying capacity for genetic material in adenoviruses than alternative viral vector platforms at around 36 kb pairs while delivering roughly 8 kb pairs. A capacity that can be increased using a newer generation of helper-dependent adenoviruses (HD-AdVs) by deleting the viral coding sequences1,37-38. Such delivery methods have been used in gene delivery to pig airway epithelia to demonstrate delivery of the CFTR protein in hopes to reverse mucus production and in�ammation in patients with cystic �brosis and associated lung diseases39. As prominent viruses in everyday life a common phenomena observed in usage of AdVs for clinical trials is pre-existing immunity 40 which presents the need for research of multiple HAdV serotypes to allow for versatility in practical usage as gene vectors. AdV types that rely on a singular method of cell entry have limited access points and see more pre-existing immunity. As previously mentioned, HAdVs types are plentiful and subcategorized into 7 grand species A-G, the majority of which belong to the HAdV-D family40. HAdV-D type 49 is one of many HAdV types that utilize varying surface molecules to enter the cell and as a result is a prime candidate for research in AdV vectors to solve this issue of immunity. The viability of HAdV-D49 is compounded by its observed transduction in lung and spleen tissues while showing reduced targeting in liver and other less desirable tissues when looking at in vivo biodistribution41. Immune response has also been mitigated by using HD-AdV vectors as their stripped down genome removes key markers for the immune system to identify and target. Airway basal cells of mice and pigs have been targeted successfully using these HD- AdVs following intranasal delivery42,43. Limiting immune response while maintaining a focus on the e�cacy of AdV based gene therapy is the current objective that needs �ne tuning in research for AdV vectors to obtain signi�cant clinical success. Adeno-associated viruses To build upon the problem of immune response many relatively distant derivatives of AdV virus vectors have been created known as adeno-associated virus (AAV) vectors. These have more desirable immunogenicity. They evoke a weak in�ammatory response compared to Berkeley Pharma Tech Journal of Medicine | 69 AdV vectors by lacking a staple viral coding sequence and contain a linear single-stranded DNA. Behavioral di�erences in host integration led to its prevalence in research as an option for gene therapy. Carrying capacity in AAV vectors is limited in comparison to AdVs at typically less than 5 kb though reports have seen up to ~9 kb of genome e�ciently incorporated into AAV vectors for therapeutics44,45. Increased packaging capacity typically comes at a cost of transduction rate and overall e�cacy in AAV vectors though research continues to be done to improve the most pressing drawback of the delivery method. Researchers at Stanford have altered charges in the lumen of AAV variants known as AAV- DJ to transduce cells derived from human kidneys better than wildtypes when packed at a higher rate of up to 6.2 kb46. More akin to lung therapies for COPD, viral/AAV chimeric gene therapy has seen signi�cant research in navigating human epithelial cells. Chimeric viral vectors look at combinations of multiple vector methods in hopes to take advantage of favorable characteristics in each and pose grounds for signi�cant advancement in gene therapy for respiratory diseases given local cell selectivity. A type of chimera vector packaging plasmids using AAV and human bocaviruses (HBoV) been shown to work well in tandem with human airway epithelia, primary human hepatocytes, skeletal muscle cells, and T cells; in addition to good performance in extensive studies in ferret airway models more recently47,48. These AAV/HBoV combination vectors o�er a novel approach to creating vectors for gene therapy in treatment of COPD. Potential Treatment There are many limitations to modern gene therapies used today caused by many biological barriers. [JG1] Most lung-related therapies must pass through multiple barriers such as the mucus, pulmonary surfactant, and local in�ammation24. This could cause a problem since patients with diseases such as asthma and cystic �brosis could also cause a biological barrier due to the airway mucus hypersecretion, which also plays a role in COPD, since around 50% of COPD patients have airway mucus hypersecretion25. Berkeley Pharma Tech Journal of Medicine | 70 Another limitation is when targeting JG3 cells other than the epithelium cells will cause the epithelium cells itself to become a major barrier24. Although there are several limitations to current gene therapies, there are other alternatives to target cells that could be considered in treatments. For example, epithelial cells, alveolar cells, and macrophages all can be accessed through inhalation of nucleic acid containing nanoparticles (NANs)26. This method of treatment helps target the lung and not target other cells that could cause biological barriers. Although inhalation of nanoparticles seems like a great treatment for lung disorders, it is actually limited in some COPD treatments. The inhalation of BIBW 2948, which is used for treating COPD, helped reduce the internalization of EFGR, which plays an important role in epithelial changes in COPD, but does not reduce the mucin storage27,28. BAMBI, which is the bone morphogenic protein and activin membrane-bound inhibitor, plays an important role in indicating if a patient has COPD. BAMBI are expressed stronger in COPD patients [28]. Also there is a correlation between the levels of BAMBI and plasma TGF -β1 levels. Therefore, the inhibition of TGF-β1 signaling might provide an alternative therapeutic strategy for treating COPD28. Inhale Gene Therapy Another potential therapeutic treatment for COPD is through Inhaled Gene Therapy. This treatment provides direct access to the target of gene therapy for obstructive lung diseases via inhalation. There are limitations that surround inhale gene therapy. In a study conducted by Dr. Magdalena Humenberger from the Kepler University Hospital in Austria concluded that complete adherence to inhaled therapy was only seen in 33.6% and was higher among those with more severe COPD, based on these results47. It was shown that mucus poses a barrier for this speci�c treatment. Although this is a current problem, other studies have mentioned a di�erent type of gene vector that could be inhaled that would penetrate that mucus barrier. A type of nanoparticle that was introduced by Berkeley Pharma Tech Journal of Medicine | 71 Dr. Jung Soo Suk, from the The Center for Nanomedicine in the Johns Hopkins University School of Medicine, was the mucus-penetrating DNA nanoparticles (DNA-MPP), which possess non-adhesive coatings that allow them to rapidly penetrate mucus layers48. The PEG coating helps with the DNA-MPP treatment since it does not have a dense surface, helping the nanoparticle to penetrate other mucin-based meshwork48. Stem Cell Therapy Another potential way of treating COPD is through stem cell therapy. This treatment is considered one of the newer treatments that still has a lot of questions �oating around it. Although stem cell therapy is not considered safe, it still has potential when it comes to treating disorders such as COPD. Recently there have been several studies and clinical trials that have focused on stem cell treatment. The studies on COPD focused on a speci�c stem cell treatment called Mesenchymal Stem Cells (MSCs), which is important for making and repairing skeletal tissues49. In a clinical study, they were infused with expanded allogeneic umbilical cord tissue derived mesenchymal stem cells (MSCs) to 20 COPD patients and then were monitored for 6 months49. This study concluded a signi�cant improvement in some important outcomes of COPD, including mMCR, CAT, number of symptoms, and the downregulation of in�ammation49. Although this study is considered a pilot study, a small scale clinical study, it provides a unique perspective when it comes to treating COPD via stem cell therapy. Alpha1-antitrypsin Alpha1-antitrypsin (A1AT) is a protease inhibitor whose de�ciency is most commonly associated with the ZZ mutation which causes abnormal folding in the ER of hepatocytes during biogenesis, causing its retention within the ER. The retention of 90% of the normal A1AT levels greatly increases the risk for early onset COPD50. In a large Lithuanian cohort, out of 1167 patients who had COPD ranging from moderate to severe, 3.4% had the MZ mutation, 3.3% had the MS mutation, 0.3% had the SZ mutation, and 0.7% had the ZZ mutations. Results from the screen indicated that there Berkeley Pharma Tech Journal of Medicine | 72 was a signi�cant increase in MZ, SZ and ZZ genotypes in COPD patients; it also supported the concept of using a targeted screen for A1AT de�ciency when diagnosing COPD51. Another study conducted in Brazil on 926 COPD patients found that 2.8% had A1AT de�ciencies and 0.8% had the ZZ mutation. These results also supported the importance of screening for A1AT levels in all COPD patients52. miRNA miRNAs play an important role in lung development, homeostasis, and pathogenesis. They also play a role in the regulation of cellular response to inhaled toxins and in regulation of in�ammatory and anti-in�ammatory processes. miRNAs are one of the primary epigenetic modi�ers that can a�ect gene expression through post-translational gene silencing and mRNA degradation. Their improper regulation can lead to chronic infections and in�ammation. A study was done that obtained lung tissue samples from 15 COPD patients and 11 subjects with normal lung function. 12 di�erentially expressed miRNA in COPD patients compared to subjects with normal lung function were identi�ed which showed to mostly target the nuclear lumen and transcription. Of the 12 di�erentially expressed miRNAs, miR-28-3p was most signi�cantly down-regulated and miR-212-5p was most signi�cantly up-regulated [53]. More recently, a study showed that two constructed miRNA-mRNA pathways; miR-126-5p and miR-130-5p-FOXO1 could be potential biomarkers for the diagnosis and treatment of COPD54. This year, a study showed that miR-126 was higher in COPD patients with acute exacerbation compared to stable COPD patients and healthy non-COPD patients and distinguished the groups. This led to the conclusion that the dysregulation of miR-126 relates to COPD susceptibility and acute exacerbation risk, but also is linked to severity and in�ammatory cytokines in COPD patients55. Berkeley Pharma Tech Journal of Medicine | 73 Discussion Chronic obstructive pulmonary disease provides unique grounds for exploration in new therapies. The compounding factors of several potential diseases or developmental miscues on a cellular level and beyond make it an issue to solve. However, this promotes much more de novo methods and research into the usage of several therapeutic methods in hopes to prevent such a prominent issue that 6.4% of Americans were diagnosed with it in 2018. Focus on lung tissue dynamics and genetic development can be accomplished in a number of ways through gene therapy is one of the most promising and has great potential in future endeavors[JG5] . As an overarching �eld, gene therapy is on the forefront of pulmonary medicine given its success in diagnosis, treatment, and identi�cation in epidemiology of cystic �brosis, emphysema, and other common issues which all act as compounding factors categorized as COPD5-9. Gene therapy works as a two pronged tool in lung therapy given the vast possibilities in the �eld between the potential delivery methods and actual genetic and cellular targets. The combination e�ect of these two issues leads to near endless possibilities in potential layouts for therapeutic strategies. In non-viral delivery methods, liposomes, nanoparticles, and electroporation are the more heavily researched aspects and provide a basis for most clinical studies1,3,15. Liposome based approaches and many potential nanoparticles limit immune response thanks to their endogenous nature and customizable size to desired scale yet the key di�erence comes when one looks at the e�ciency. Liposomes and liposomal NPs sacri�ce ease of use and optimal immunogenicity for e�ciency in many cases due to the body naturally developing barriers to them such as nuclear transport of their genetic material and signi�cant degradation before they can have a signi�cant e�ect on the target15,17. Non-liposomal NPs are the most wide- open area of research given the plethora of potential formulations. PLGA and dendrimer nanoparticles for lung therapy have seen success given their idealized interaction. Yet, price points become an issue when one looks at the extensive research and time that must be put into each individual iteration of a given treatment method22,30. For example, Berkeley Pharma Tech Journal of Medicine | 74 taking a simple PLGA coated nanoparticle with targeting sites to deliver to lung epithelia for production in A1AT vs. the exact same base particle complemented to target ciliated cells will require entirely di�erent sets of trials and FDA compliance before any signi�cant levels of usage can occur. This is obviously necessary for safety and ethical concerns but presents a signi�cant holdup in advancement of NP as a therapy for COPD. Electroporation uses the manipulated electric �elds in cells to increase permeability signi�cantly. Targeting speci�city and overall quality of research eludes the �eld but it presents a purely physical method for gene therapy delivery and even assisting other methods should cell access present a signi�cant issue1,33. Viral methods for delivering gene therapy are composed of mostly adeno and adeno-associated viruses given the broad spectrum they o�er for behavior and ease of modi�cation35. Both o�er one of the more sought after qualities in gene therapy strategies in their integration abilities. The inherent function of viruses makes them ideal vectors that just need to be altered for the desired function. Immune response is an issue that can be improved upon but presents one of the only signi�cant issues in their usage as gene vectors44. Targets for the readily available delivery methods is where the speci�city of COPD comes to the forefront of treatment. Inhalable therapies provide a unique avenue for treating pulmonary issues that might not be possible with other groups of diseases, thus it has been explored heavily with iterations of liposomal NP and viral vector bodies optimized for breaking through the heavy mucus and surfactant layers in lung tissues17,42-43,48. A variety of cells are still accessible through this minimally invasive gene therapy26. New stem cell therapy for COPD focus primarily on mesenchymal stem cells given their limited exhibition of pluripotent properties. One of the most well researched causes and potential targets for COPD is that of A1AT where gene alteration to the common ZZ mutation or to improve production in A1AT de�cient individuals has been seen to improve both physiological qualities and provided long term expression50-52. The �nal but potentially most broad method for focusing on the issues presented in COPD is that of working with miRNA using the aforementioned delivery methods. As a primary regulator for gene expression, in depth studies have found disparities in miRNA quality in Berkeley Pharma Tech Journal of Medicine | 75 COPD patients53. Insertion of NPs to change miRNA expression for issues like lung irritation have also seen success56. Looking at these delivery methods in tandem with targeting goal is the only way to e�ectively evaluate the viability of a therapeutic strategy for COPD and look to improve the current state of understanding in treatment for it. Conclusion Although there are many potential treatment avenues, there needs to be a large focus on personalized medicine as the source of patients’ COPD can vary. Severity and associated symptoms are plentiful in COPD which only serves to necessitate more research into the �eld. Making strides towards being able to identify the best suited treatment for an individual will result in the most e�ective outcomes and treatments. 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