key: cord-0998438-x9vhhb9v authors: Chow, MichaelY.T.; Chang, Rachel Yoon Kyung; Chan, Hak-Kim title: Inhalation delivery technology for genome-editing of respiratory diseases date: 2020-06-05 journal: Adv Drug Deliv Rev DOI: 10.1016/j.addr.2020.06.001 sha: 04026ce0967dc62fc1853568f88bc2374c35aaf2 doc_id: 998438 cord_uid: x9vhhb9v The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has significant therapeutic potentials for lung congenital diseases such as cystic fibrosis, as well as other pulmonary disorders like lung cancer and obstructive diseases. Local administration of CRISPR/Cas9 therapeutics through inhalation can achieve high drug concentration and minimise systemic exposure. While the field is advancing with better understanding on the biological functions achieved by CRISPR/Cas9 systems, the lack of progress in inhalation formulation and delivery of the molecule may impede their clinical translation efficiently. This forward-looking review discussed the current status of formulations and delivery for inhalation of relevant biologics such as genes (plasmids and mRNAs) and proteins, emphasising on their design strategies and preparation methods. By adapting and optimising formulation strategies used for genes and proteins, we envisage that development of inhalable CRISPR/Cas9 liquid or powder formulations for inhalation administration can potentially be fast-tracked in near future. CRISPR/Cas9 has been widely viewed as the preferred candidate for its superior efficiency and specificity, which also comes with a lower cost and does not require dedicated enzyme engineering [1, 2] . Originated from bacterial adaptive immune system [3] , the CRISPR/Cas9 system comprises a Cas9 endonuclease and a guiding CRISPR RNA (crRNA) to which a trans-activating crRNA (tracrRNA) binds and forms an activated complex. These two short RNAs can also be optimised and combined into one single-guide RNA, or sgRNA. The Cas9 endonuclease then recognises specific region of the target DNA as guided by the sequence of the crRNA, resulting in DNA cleavage or double-strand breaks [4] . Depending on the usage, the cellular DNA repair mechanism can undergo non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ is error-prone that can result in small insertions or deletions (indels) in target sites, causing interruption or elimination of the function of the cleaved genes. In contrast, HDR utilises a homologous repair template to achieve high fidelity genome correction, which can potentially correct innate disease-causing errors of genes. The utility of CRISPR/Cas9 system was initially recognised as a valuable and powerful research tool to understand the casual roles of specific genetic variations, instead of relying on disease models where only a particular disorder is phenocopied [5] . Like other genetic modification techniques such as RNA interference, the potential of this precise genome editing technology as a novel therapeutic strategy has been explored in many diseases, including pulmonary disorders. Majority of lung congenital diseases, such as cystic fibrosis (CF) and α-1 antitrypsin deficiency, are caused by monogenetic mutation, rendering the use of precision genomeediting tool like CRISPR/Cas9 an attractive idea. Moreover, the high prevalence of lung cancers and refractory obstructive respiratory diseases like asthma and chronic obstructive pulmonary disorder remains an enormous socioeconomic burden to even the more developed countries, representing a gargantuan unmet medical need [6] [7] [8] . For further details on genome-editing biomacromolecules for lung genetic disorders, the readers are referred to the article by Wan and Ping in this theme issue [9] . Pulmonary delivery of therapeutic genes by inhalation demands the biopharmaceuticals to be Intravenously injected genetic materials are distributed to different organs in the body such as kidney, liver and spleen [16] . They also reach and accumulate in the alveolar region rather than the ciliated epithelial cells in the bronchi. An implication of which is the potential limitation of intravenous administration in treating some pulmonary genetic disorders such as CF, since cystic fibrosis transmembrane conductance regulator (CFTR), the mutation of which causes CF, is primarily expressed in bronchi. Some of these challenges can be partially mitigated by the local administration of geneediting therapeutics as aerosols through oral inhalation to the lungs. It enables genes, either in its naked form or complexed with delivery vectors, to be delivered directly and rapidly to the target cells in the lungs at a high concentration with reduced systemic exposure [17] . Local delivery also allows non-invasive access to the lungs and minimises the interactions between genes and serum proteins [18, 19] . In fact, the large alveolar area with high vascularisation, coupled with its thin air-blood-barrier [20] and relatively low enzymatic activity, also render pulmonary administration a viable alternative for systemic treatments. In spite of these J o u r n a l P r e -p r o o f advantages, the development of inhalable genome-editing therapeutics has been relatively slow due to the complexity involved. For drugs to be administered through inhalation, they must be formulated into either a liquid or solid aerosol. It follows that the aerosol properties of the formulations need to be precisely controlled for efficient delivery aerosols to the lungs. In pharmaceutics, one of the most relevant measurements of aerosol properties is aerodynamic diameter. The aerodynamic diameter of a single particle is defined as the physical (geometrical) diameter of a spherical particle with density of 1 g cm -3 that has the same settling velocity in air as the particle in question. It is well known that in general inhaled particles with aerodynamic diameter of 1 to 5 µm are suitable for respiratory delivery, whereas lung deposition decreases outside this range either due to impaction loss of large particles in the oropharynx or exhalation of small particles, depending on the inspiratory flowrate and pausing [15, 21] . In patients with obstructive airway diseases such as CF, aerosols with aerodynamic diameter of 2 to 3 µm is considered to provide the greatest therapeutic benefit [22] . The aerodynamic size distribution of an aerosol (i.e. a collection of individual particles) can be measured by cascade impactors in tandem with suitable dispersion devices such as nebulisers or dry powder inhalers (DPI). Cascade impactors segregate particles according to their aerodynamic diameters through the principle of inertial momentum. There are two main derived statistics of aerodynamic size distribution, namely the mass median aerodynamic diameters (MMAD) and fine particle fraction (FPF). MMAD refers to the aerodynamic diameter under (and also above) which half In evaluating the aerosol performance of formulations for inhalation, it should be emphasised that in vivo animal models have limited utilities for such purpose due to significant anatomical and physiological differences between species. Moreover, the different methodologies for pulmonary drug delivery to animals each possess their own strengths and limitations [26] . For example, aerosol administration via passive inhalation such as wholebody exposure or head-/ nose-only exposure systems spare the animal from anaesthesia, yet an accurate control over the delivered dose can be difficult to achieve. Intratracheal administration allows the delivery of a controlled amount of drug, but it might be technically more demanding particularly in smaller animals such as mice. Given these limitations, in vivo animal models should be primarily used to demonstrate the safety, efficacy and possibly pharmacokinetics of the formulations. In addition to aerodynamic diameter, other particle physicochemical properties such as size, charge, density and surface composition can influence the fate of drug deposition and cell uptake in the lungs [22] . Furthermore, a therapeutic gene formulation must remain inhalable, with sufficient stability and aerodynamic properties during storage and use [27] . Only through the intricate engineering of the biomolecules, excipients in the formulation and the delivery device can a safe and efficient delivery be achieved, yet the final clinical outcomes of which is also inevitably affected by the lung pathophysiology of the patients. As only a limited number of studies in this field has been reported, the following sections will primarily leverage on the prior art in inhaled gene therapy and its potential application for the aerosol delivery of CRISPR/Cas9-based platforms. J o u r n a l P r e -p r o o f Despite numerous advantages afforded by pulmonary administration, successful aerosol delivery of biopharmaceuticals including CRISPR/Cas9-based therapeutics remains highly challenging. There exist several major anatomical and physiological barriers that prevent the successful delivery genome editing via inhalation to the lungs [19, 28] . Firstly, oropharyngeal deposition of orally inhaled aerosol particles must be avoided to maximis e lung deposition. The highly bifurcated anatomy of the lung together with the constricting airway down the respiratory tract serve as an effective structural barrier to prevent penetration of large particles to the lower respiratory tract [21] . Secondly, the mucus lining secreted by the epithelia has been considered as another major barrier in aerosol delivery. Airway mucus is primarily composed of a gel-type mucin fibre that contains a high density of negatively charged macromolecules [29] . As most nanocomplexes of genes and delivery vectors are cationic, they are prone to accumulation in the mucus layer via a manifold of interactions. These interactions, including hydrophobic forces, hydrogen bonding and electrostatic interactions, not only impair the colloidal stability of vectors and reduce their delivery efficiency, but also make the gene molecules in the cargo vulnerable to degradation by the surrounding nucleases [29] . Thirdly, the situation is further complicated by the disease state, for instance, patients suffering from CF have an accumulation of abnormally thick and stick mucus due to the lack of CFTR functions. Their airways are often found inflamed and infections are commonplace. As a result of the constricted airways, turbulent flow is enhanced which reduces the average travel distance of aerosol particles. Aerosol particle deposition is hence affected as it is more probable for the particles to deposit earlier on large airways by local turbulence [30] , making peripheral deposition less favourable [31] . Paradoxically, diseased regions where drug particles should deposit would also receive less air flow and thus a lower dose as a result of obstruction. Prediction of deposition patterns of J o u r n a l P r e -p r o o f inhaled genes in lungs of CF patients became less accurate since the airways can differ significantly, depending on the disease states and resulting complications. As stated above, the viscous and dense mucus also act as formidable barriers, further hindering the gene uptake by target epithelial cells [32] . collapsed and their complexation is disrupted [34, 35] . Fifthly, alveolar macrophages residing in the airspace ( Figure 1 ) also constitute yet another barrier that hampers gene delivery through inhalation [36] [37] [38] [39] . Several intracellular barriers, such as endosomes and the nuclear envelope, must be overcome during gene delivery after internalis ation by the target cells through the judicious use of delivery vectors [40] , of which the latest development for CRISPR/Cas9 has been thoroughly reviewed [2, [41] [42] [43] [44] [45] [46] . Although non-viral vectors often remain inferior to viral vectors in their transfection efficiency at the trade-off for a safer immunogenic profile, systematic and in-depth study on their structure-function relationships can bring about engineered vectors with improved efficacy [47] . Strategies to overcome these physiological barriers for inhaled gene therapy, such as modification of delivery vectors and modulations of barriers, have been proposed and discussed in detail [19, 48] . The incorporation of specific ligands to the vector surface by J o u r n a l P r e -p r o o f covalent conjugation or genetic engineering has been widely explored to reduce adhesive interactions with mucus, enhance particle stability, and reduce macrophage uptake, which further endow viral and non-viral vectors the ability to be efficiently internalised by target cells [49] [50] [51] [52] [53] [54] . The use of mucus-altering agent, including N-acetyl cysteine and recombinant human DNase, can increase mucus mesh pore size, thereby facilitating the penetration of gene nanocomplexes through the mucus layer [55, 56] . Meanwhile, the incorporation of osmotic agents to inhaled formulations, such as the use of hypertonic saline and mannitol, can increase mucus and/or PCL mesh pore size by hydration and thus improve mucociliary clearance [57, 58] . Tight junctions disrupting agents, such as sodium caprate, polidocanol and lysophosphatidylcholine, can transiently disrupt tight junctions in the epithelial layer to provide an access to the basolateral surface for gene vectors [59, 60] . These well-established strategies will facilitate the rational design of aerosol inhalation delivery for genome-editing agents. J o u r n a l P r e -p r o o f Unlike other routes of administration, pulmonary administration consists of not only the drug formulation but also a suitable inhaler device. Nebuliser, pressurised metered dose inhaler (pMDI) and DPI are the three major types of inhaler device that can disperse liquid (nebuliser and pMDI) or solid (DPI) into inhalable aerosols. Among these devices, pMDI is a common device to deliver potent small molecules like beta adrenergic agonists, inhaled cortical steroids and anticholinergics for the management of obstructive lung diseases. Apart from some early investigations [61] [62] [63] , studies on the delivery of biopharmaceutical by pMDI has Vibrating mesh nebulisers generate less heat in the liquid, thus reducing the potential for thermal or chemical degradation. However, nebulisers based on ultrasonic vibration may not be powerful enough to aerosolise formulations of pDNA polyplexes even at a moderate concentration of 0.2 mg/ml, because of high liquid viscosity (at 6.3 ± 0.1 cP, whereas water has a viscosity of 1 cP at 20°C) [68] . Presence of large aggregates in the formulation may also hinder liquid transfer through the micron-sized orifices of vibrating-mesh nebulisers [69] . Regardless of the nebulisation principle, in situ degradation during aerosolization remains a major formulation consideration for nebulisation, as biopharmaceuticals including CRISPR/Cas9 therapeutics are prone to degradation when exposed to hydrodynamic shear J o u r n a l P r e -p r o o f stress [70] [71, 72] and in vivo [73, 74] . These earlier studies emphasised more on the effects of different nebulisation processes on the transfection efficiency of the pDNA lipoplexes. It was shown that the efficiency of transfection decreased over the course of nebulisation, with only about 25% to 35% of initial activity remained at 10 minutes for both the Aerotech II and the Puritan-Bennett 1600 nebulisers [71] . The degradation could be partially mitigated by increasing reservoir volume or decreasing flow rate. With optimisation of the ratio between the cationic lipid carrier and pDNA, it was possible to maintain the integrity of the complex over the nebulisation process [74] . Manunta et al. reported the nebulisation of nanocomplexes comprising cationic liposomes containing pDNA and receptor-targeting peptides, named as receptor-targeted nanocomplex (RTN) [75] . A total of three nebulisers were tested, and the RTN system was most effective in terms of transfection efficiency when nebulised by the AeroEclipse® II BAN air-jet nebuliser. The deposition pattern of the RTN aerosol in pigs was then visualised using technetium-99m labelled radiopharmaceuticals [54] . It was found that the nebulised radiovectors were primarily deposited in the trachea-main bronchi and in the middle region of the lungs, with the scintigraphy images correlating to the plasmid biodistribution. In a study comparing the performance of four different nebulisers, the breathactuated AeroEclipse® II nebuliser was also selected as the preferred device in the clinical J o u r n a l P r e -p r o o f studies of the pulmonary delivery of the cationic lipid GL67A-conjugated pGM169 plasmid, a CFTR gene therapy in patients with CF [76, 77] . Research on formulations for ultrasonic nebulisers also yielded some success [78, 79] [14] . In this study, poly(ethylene glycol) (PEG) monomethyl ether with different molecular weights was conjugated chitosan. Chitosan is a natural polymer that has been widely used as a non-viral delivery vector because it is biocompatible, biodegradable and is generally non-toxic even in large doses [83] . Its mucoadhesive property has been utilised in oral and nasal gene delivery for vaccination [84] . However, this property has become a disadvantage for pulmonary gene delivery especially in Table 1 . Pulmonary delivery of therapeutic proteins has always been of high therapeutic interest primarily because of its non-invasiveness and reduced degradation activity, when compared with other administration routes such as systemic injection and oral administration [88] . Dornase alfa, a recombinant human DNase indicated for patients with CF, was the first and only protein being approved by pulmonary delivery for local therapeutic effects in the lungs. Since its use in 1993 as an adjuvant therapy, the qualities of life of the patients have been drastically improved. Currently, more than 18 inhaled protein therapeutics have reached clinical trials, with indications on both major respiratory disorders such as asthma and pneumonia and rare or orphan lung diseases like CF and alpha-1 antitrypsin deficiency [65] . While the design of formulations suitable for nebulisation of proteins is highly protein specific [89] , the formulations can remain relatively simple. By utilis ing surface acoustic waves, simple aqueous solution of epidermal growth factor receptor (EGFR) monoclonal antibodies at 200 µg/ml can be readily nebulised into fine aerosols with a mass median aerodynamic diameter of about 1.1 µm as measured by the NGI [90] . The protein integrity after nebulisation was evaluated qualitatively using gel electrophoresis. Flow cytometry with A549 cell lines showed that the antigen binding capability of the antibody was retained, and phosphorylation in A431 human epidermoid carcinoma cells overexpressing EGFR was reduced. On the other hand, commercially available preparation of interferon-γ for injection (Imukin® from Boehringer Ingelheim) could be directly nebulised using two types of vibrating mesh nebuliser without sacrificing structural integrity and bioactivity [91] . The post-nebulisation stability of the protein was assessed by both gel electrophoresis and in vitro J o u r n a l P r e -p r o o f assay. The overall strategies employed in stabilis ing monoclonal antibodies may well be adapted and optimised for Cas9 nuclease formulations [92] . Surfactants act through replacing protein molecules at the air-liquid interface during nebulisation [93, 94] . Sugars, such as trehalose and mannitol, as well as polyethylene glycols (PEG) stabilise proteins by steric hindrance, hence preventing protein aggregations [65] . The role of amino acids in improving stability of protein formulations has also been well recognised, although the actual stabilis ing mechanisms remain to be elucidated [95] . Whilst nebulisation, due to its simple formulation design, remains the obvious choice for many scientists utilis ing genome-editing as a research tool to study genetic disorders or These biopharmaceuticals also differ from small potent molecules such as inhaled cortical steroids or bronchodilator that they have to be administered at high drug doses (often in milligrams), potentially necessitating high-performance powder formulations and inhalers [99] [100] [101] . As in liquid aerosols, the dry powders must also exhibit proper aerodynamic and physical properties for them to be suitable for inhalation, which is further complicated by the scant excipients approved for use in formulations for pulmonary administration. Lastly, these biomolecules are often costly to manufacture and thus a cost-effective manufacturing process with high recovery yield is highly preferred. Among the different techniques for manufacturing inhalable powders of biopharmaceuticals, spray drying (SD), spray freeze drying (SFD) and supercritical fluid (SCF) drying are the three methods that have been mostly investigated [102, 103] . SD involves atomising a feed solution into a fine spray followed by drying of droplets in one single continuous step. Different process parameters can be fine-tuned to achieve the desirable particle attributes. As standardised spray dryers are available at various production scales, spray drying represents a popular drying process to pharmaceutical manufacturing [104] , including inhalation products of insulin and mannitol. SFD consists of atomisation of the feed solution into a cryogen, followed by lyophilisation of the frozen droplets [105] . pDNA conjugated with a non-viral lipid-polycation delivery vector [106] . Subsequently, the effects of non-condensing sugars [107] or amino acids [108] [109] [110] on improving the spray dried powder dispersibility and pDNA stability have been reported. During the same period, Okamoto et al. in Japan began their investigation on employing SCF drying to prepare inhalable dry powder of pDNA. They reported the preparation of chitosan-pDNA complex powders by SCF drying [111] , and later confirmed their improved pDNA stability during manufacturing and storage for up to 4 weeks [112] . The in vivo pulmonary delivery and gene expression upon the administration of the pDNA powders in mice was then evaluated using fluorescent label pDNA [113] . On the other hand, while the potential of preparing pDNA dry powder by SFD has been briefly explored by Kuo et al. in 2004 [114] , it was not until several years later when Mohri et al. attempted to develop and optimise the formulations of chitosanconjugated pDNA [115] . Subsequent investigations focused on studying the effect of bovine serum albumin and the amino acid leucine as lyoprotectant during freeze drying [116, 117] , as well as the use of alternative biodegradable polycation as the transfection vector [118] . hyaluronic acid (LHA) as an excipient [120] . Possible reasons included the presence of a specific intracellular uptake mechanism via a receptor, and LHA exhibits favourable inhalation characteristics as an excipient for inhalation powder. The storage stability of this formulation in terms of aerosol performance and gene expression has also been recently demonstrated [121] . The aerosol performance, the structural integrity and the in vivo gene expression activity of the powders under 25 °C and a dry condition were found to be stable over a storage period of up to 12 months. As efficient and safe gene delivery vectors are still much sought-after, the abolishment of transfection agents desirably eliminated their associated toxicities. The other form of CRISPR/Cas9 therapeutics is the administration of mRNA that encodes Cas9 nucleases. In contrast to pDNA, only limited studies on formulating RNAs into dry powders for inhalation have been reported. Most of these studies investigated powder formulations of short interfering RNA (siRNA), a non-coding double-stranded RNA consisting 20-25 base pairs that silences gene expression through RNA interference (RNAi) [122, 123] . Inhalable siRNA dry powders have been successfully engineered using SD [124] [125] [126] [127] [128] , SFD [129] [130] [131] [132] or SCF drying techniques [133] . powder by both SD and SFD [134] . PEGylated synthetic cationic KL4 peptides were used as the transfecting agent through electrostatic interactions with the negatively charged mRNAs. While there was no specific optimisation on the drying conditions, by adopting the operation parameters developed for siRNA formulations [124, 129] , the bioactivity of the peptide/mRNA complexes were preserved upon drying using both techniques. Using a luciferase mRNA as the model gene, intratracheal administration of the peptide/mRNA complexes dry powder resulted in luciferase expression in the deep lung region of mice 24 hours post transfection. The powders also exhibited satisfactory aerosol performance, demonstrating a fine particle fraction (fraction of particles with aerodynamic diameters of 5 µm or below) of 41% and 68% for the spray dried powders and the spray freeze dried powders, respectively. Importantly, the powder formulations did not show signs of inflammation in the lungs. This study served as a milestone in demonstrating the feasibility and potential of formulating mRNA therapeutics into inhalable dry powders. Delivery vectors, excipients and preparation methods used to prepare powder formulations of plasmid DNAs and mRNAs which are relevant for pulmonary delivery of CRISPR/Cas9 were summar ised in Table 2 . Similar to genes, the pulmonary delivery of proteins in dry powder form has been of great interest since it allows both the local treatment of respiratory disorders or as a non-invasive route to systemic drug delivery and elimination of risks of biochemical instability in liquid formulations [135, 136] . Earlier investigations include the preparation of recombinant human granulocyte-colony stimulating factor (G-CSF or filgrastim) [137] and recombinant human deoxyribonuclease (rhDNase or dornase alfa) [138] [139] [140] [145] . The effect of operating conditions, formulation excipients and subsequent processing conditions on protein stability, powder morphology, powder aerosol performance and powder residual moisture has been evaluated [146] [147] [148] [149] [150] . Amino acids [151, 152] , sugars [153] [154] [155] [156] [157] [158], surfactants [88, 159] , cyclodextrin [160, 161] and polymers [162] are examples of excipients used to promote protein stability and improve powder characteristics. Faghihi et al. characterised and optimised an inhalable powder formulation of IgG antibodies by a design of experiment approach on the levels of three excipients (cysteine, trehalose and tween 20) [163] . Utilis ing an ovalbumin-challenged mice model, the group later demonstrated that the bioactivity of infliximab was preserved upon spray drying as reflected by a reduced TNFα (tumour necrosis factor alpha) secretion in lung tissues [164] . As an alternative to SD, SFD J o u r n a l P r e -p r o o f has gained popularity recently [157, 161] . Monoclonal antibodies like omalizumab and adalimumab has been formulated into inhalable dry powders by SFD technique [145, 152] . The presence of amino acids leucine or phenylalanine helped retaining adalimumab stability during SFD and over a 3-month storage under accelerated conditions. Excipients and preparation methods used to prepare dry powder formulations of protein were summarised in Table 3 . To date, there has been no published study on the preparation of Cas9 nuclease into inhalable dry powders. Cas9 nuclease has a bilobed structure comprising a target recognition lobe and a nuclease lobe [165] , and is morphologically different to monoclonal antibody that consists of two pairs of light chains and heavy chains in an Y-shape formation. The size of Cas9 nuclease (from Streptococcus pyogenes, SpCas9) is 162 kDa, and is larger than that of monoclonal antibodies which typically ranges between 140 to 150 kDa. While the optimal formulation design in fabricating proteins into inhalable powders is largely dependent on the physicochemical properties of the proteins, existing knowledge and experience with monoclonal antibodies can still shed some light on formulating Cas9 nuclease into powder formulations for pulmonary administration. For instance, functional excipients to enhance the physical and chemical stability of amorphous proteins can be harnessed to improve the properties of the powder formulations for Cas9 [166] . Such extrapolation of engineering strategies is also expected to be applicable for formulations of pDNAs and mRNAs. Critically, the distinction of the physicochemical properties, such as size and zeta potential, between the CRISPR/Cas9 cargoes or the cargoes-delivery vector assemblies, and those that have been reported on other biomolecules must be observed and rational adaptation has to be exercised. The size of CRISPR/Cas9 cargoes, as well as model genes and proteins that are commonly used for formulation development in the aforementioned formulation studies are summarised in Table 4 . Most of the model biomolecules used for formulation development have a shorter J o u r n a l P r e -p r o o f sequence than the CRISPR/Cas9 cargoes, rendering the formulation of the latter more challenging. J o u r n a l P r e -p r o o f Inhalation delivery of CRISPR/Cas9 as an aerosol is a manifold challenge, demanding a holistic consideration and engineering in the CRISPR/Cas9 cargoes and the accompanied delivery vector, the formulation and its manufacture, the dose required and the aerosol performance and biochemical stability (including long-term storage) of the delivery platform. Undoubtedly, these factors substantially interact with each other, and the selection of them will be affected or dictated by other factors, depending on the priorities of the formulation. For instance, the dose of the CRISPR/Cas9 therapeutics is dependent on the cargoes used, and in turn by the extent of control over Cas9 expression one has to achieve, as cargoes that rely on cellular transcription will result in a less predictable Cas9 activity compared to direct administration of nuclease. The dose will also be affected by the delivery efficiency of the vectors and will be limited by the capacity (such as the extent of mechanical stress exerted on the biomolecules during manufacturing / aerosolization and the drug payload) of the delivery platform. However, inhalation formulation and delivery technology has advanced rapidly over the past 25 years. What have been learned about aerosol delivery of biological molecules such as genes and proteins can be adapted and optimised for delivery of CRISPR/Cas9 therapeutics. A proof-of-concept study of CRISPR/Cas9 has already confirmed the feasibility of its delivery by nebulisation [14] . As the potential in CRISPR/Cas9 genome-editing tool has just been unveiled, its applications for pulmonary diseases are expected to expand. There are already investigations with propitious outcomes on using CRISPR/Cas9 for re-sensitis ing bacteria to resistant antibiotics [167, 168] , for use as a direct antibacterial agent [169] or for modifying and improving the host range and bactericidal activity of bacteriophages [167] . This opens a new research space for scientists to attempt many pulmonary delivery strategies involving solution formulation properties, [79] gWIZ™ GFP, Naked Omron MicroAIR® NE-U22 [80] J o u r n a l P r e -p r o o f pQR150 pEGFP-N1 DHDTMA/DOPE AeroEclipse II BAN [54, 75] pVR1020 Naked Surface acoustic wave nebuliser [82] pCFTR GL-67A Various, AeroEclipse II BAN [68, 76] pSpCas9 PEGylated chitosan Aerogen Solo [14] pEGFP Cell penetrating peptide Omron MicroAIR® NE-U22 [81] mRNA MetLuc, eGFP DMRIE/cholesterol Pari Boy nebuliser [85] Luciferase hPABEs Aerogen AeroNeb [87] DC pCMV-Luc PEI. PEG Sucrose SD [107] gWIZ™ Luc pH responsive peptides Mannitol SD, SFD [119] pCAG-Luc PAsp(DET), PEG-Leucine, mannitol SFD [117, 118] J o u r n a l P r e -p r o o f PAsp(DET) pCpG-ΔLuc Naked Hyaluronic acid SFD [120, 121] mRNA Luciferase PEG-KL4 peptide Mannitol SD, SFD [134] DOTAP Figure 1 . Schematic representation of physiological barriers to pulmonary CRISPR/Cas9 delivery. Particles must first avoid deposition in the oral cavity and at the oropharynx (1) before reaching the airway. Those deposited on the airway epithelium are under the constant elimination by mucociliary clearance (2) . Biopharmaceuticals in the particles must penetrate through the surfactant, the mucus lining and periciliary layer that consist of a meshwork of cell-tethered mucins (3) before they can enter pneumocytes. Macrophages are present in alveola to phagocytise foreign particles (4). 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Part I: physiological factors affecting therapeutic effectiveness of aerosolized medications Evaluation and modification of commercial dry powder inhalers for the aerosolization of a submicrometer excipient enhanced growth (EEG) formulation Dry powder inhalation: past, present and future Degree of throat deposition can explain the variability in lung deposition of inhaled drugs Preclinical models for pulmonary drug delivery Inhalation delivery of complex drugs-the next steps Rational particle design to overcome pulmonary barriers for obstructive lung diseases therapy Barrier properties of mucus Aerosol deposition in health and disease Effect of cystic fibrosis on inhaled aerosol boluses Pulmonary drug delivery: from generating aerosols to overcoming biological barriers-therapeutic possibilities and technological challenges A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia Pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro Pegylated GL67 lipoplexes retain their gene transfection activity after exposure to components of CF mucus Internalization of adenovirus by alveolar macrophages initiates early proinflammatory signaling during acute respiratory tract infection Proteomics of bronchoalveolar lavage fluid Parameters influencing the stealthiness of colloidal drug delivery systems Influence of particle size on drug delivery to rat alveolar macrophages following pulmonary administration of ciprofloxacin incorporated into liposomes Chemical vectors for gene delivery: uptake and intracellular trafficking Genetic therapies for cystic fibrosis lung disease Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field Delivery strategies of the CRISPR-Cas9 geneediting system for therapeutic applications Recent advances in aerosol gene delivery systems using non-viral vectors for lung cancer therapy Cancer gene therapy: innovations in therapeutic delivery of CRISPR-Cas9 Chitosan in Non-Viral Gene Delivery: Role of Structure, Characterization Methods, and Insights in Cancer and Rare Diseases Therapies Structurefunction relationships of nonviral gene vectors: Lessons from antimicrobial polymers Enhancement of lung gene delivery after aerosol: a new strategy using non-viral complexes with antibacterial properties Lung gene therapy with highly compacted DNA nanoparticles that overcome the mucus barrier Use of singlesite-functionalized PEG dendrons to prepare gene vectors that penetrate human mucus barriers Targeting the urokinase plasminogen activator receptor enhances gene transfer to human airway epithelia Entrainment of neuronal oscillations as a mechanism of attentional selection Interaction between Neuronal Depolarization and MK-801 in SH-SY5Y Cells and the Rat Cortex Airway deposition of nebulized gene delivery nanocomplexes monitored by radioimaging agents Reduction in viscoelasticity in cystic fibrosis sputum in vitro using combined treatment with nacystelyn and rhDNase Rapid transport of muco-inert nanoparticles in cystic fibrosis sputum treated with N-acetyl cysteine Mucus clearance and lung function in cystic fibrosis with hypertonic saline Inhaled mannitol improves the hydration and surface properties of sputum in patients with cystic fibrosis Enhancement of adenovirus-mediated gene transfer to the airways by DEAE dextran and sodium caprate in vivo Safety and efficiency of modulating paracellular permeability to enhance airway epithelial gene transfer in vivo Novel pMDI formulations for pulmonary delivery of proteins In vitro reporter gene transfection via plasmid DNA delivered by metered dose inhaler Propellant-based inhalers for the non-invasive delivery of genes via oral inhalation Nanoparticle-Based Delivery of CRISPR/Cas9 Genome-Editing Therapeutics Heuze-Vourc'h, Designing inhaled protein therapeutics for topical lung delivery: what are the next steps? Aerosol Delivery Devices for Obstructive Lung Diseases Formulations and nebulizer performance Aerosol delivery of DNA/liposomes to the lung for cystic fibrosis gene therapy New aerosol delivery devices for cystic fibrosis Effect of jet nebulization on DNA: identifying the dominant degradation mechanism and mitigatio n methods Delivery of DNA-cationic liposome complexes by small-particle aerosol Simulated lung transfection by nebulization of liposome cDNA complexes using a cascade impactor seeded with 2-CFSME0-cells Optimization of formulations and conditions for the aerosol delivery of functional cationic lipid:DNA complexes Aerosolization of cationic lipid:pDNA complexes--in vitro optimization of nebulizer parameters for human clinical studies Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind A randomised, double-blind Ultrasonic nebulization of cationic lipid-based gene delivery systems for airway administration Aerosolization of cationic lipid-DNA complexes: lipoplex characterization and optimization of aerosol delivery conditions Studies on aerosol delivery of plasmid DNA using a mesh nebulizer Delivery of pDNA Polyplexes to Bronchial and Alveolar Epithelial Cells Using a Mesh Nebulizer Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization Natural polymers for gene delivery and tissue engineering Chitosan IFN-gamma-pDNA Nanoparticle (CIN) Therapy for Allergic Asthma Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration Interaction of polyamine gene vectors with RNA leads to the dissociation of plasmid DNA-carrier complexes Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium Protein stability in pulmonary drug delivery via nebulization Antibody structure, instability, and formulation Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform Effective nebulization of interferon-γ using a novel vibrating mesh Heuze-Vourc'h, Nebulization as a delivery method for mAbs in respiratory diseases Heuze-Vourc'h, Effect of formulation on the stability and aerosol performance of a nebulized antibody Prediction of protein degradation during vibrating mesh nebulization via a high throughput screening method Inhaled proteins: challenges and perspectives Exhaled air and aerosolized droplet dispersion during application of a jet nebulizer Severe acute respiratory syndrome (SARS): lessons learnt in Hong Kong Severe acute respiratory syndrome (SARS) and healthcare workers Dosing challenges in respiratory therapies The Delivery of High-Dose Dry Powder Antibiotics by a Low-Cost Generic Inhaler High dose dry powder inhalers to overcome the challenges of tuberculosis treatment Dry Powder Formulation of Plasmid DNA and siRNA for Inhalation Drying Technologies for the Stability and Bioavailability of Biopharmaceuticals Spray-dried powders for pulmonary drug delivery Spray freeze drying: Emerging applications in drug delivery Preparation of dry powder dispersions for non-viral gene delivery by freeze-drying and spray-drying The effect of protective agents on the stability of plasmid DNA by the process of spray-drying Enhanced dispersibility and deposition of spray-dried powders for pulmonary gene therapy The use of amino acids to enhance the aerosolisation of spray-dried powders for pulmonary gene therapy The use of absorption enhancers to enhance the dispersibility of spray-dried powders for pulmonary gene therapy Pulmonary gene delivery by chitosan-pDNA complex powder prepared by a supercritical carbon dioxide process Stability of chitosan-pDNA complex powder prepared by supercritical carbon dioxide process Dual imaging of pulmonary delivery and gene expression of dry powder inhalant by fluorescence and bioluminescence Preparation of DNA dry powder for non-viral gene delivery by spray-freeze drying: effect of protective agents (polyethyleneimine and sugars) on the stability of DNA Optimized pulmonary gene transfection in mice by spray-freeze dried powder inhalation Bovine serum albumin as a lyoprotectant for preparation of DNA dry powder formulations using the spray-freeze drying method Development of New Formulation Dry Powder for Pulmonary Delivery Using Amino Acids to Improve Stability Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids Naked pDNA Inhalation Powder Composed of Hyaluronic Acid Exhibits High Gene Expression in the Lungs Naked pDNA/hyaluronic acid powder shows excellent long-term storage stability and gene expression in murine lungs Delivery of RNAi Therapeutics to the Airways-From Bench to Bedside siRNA Versus miRNA as Therapeutics for Gene Silencing Inhaled powder formulation of naked siRNA using spray drying technology with l-leucine as dispersion enhancer Inhalable siRNA-loaded nano-embedded microparticles engineered using microfluidics and spray drying Design of an inhalable dry powder formulation of DOTAP-modified PLGA nanoparticles loaded with siRNA Spray drying of siRNA-containing PLGA nanoparticles intended for inhalation High siRNA loading powder for inhalation prepared by co-spray drying with human serum albumin Using two-fluid nozzle for spray freeze drying to produce porous powder formulation of naked siRNA for inhalation Development of spray-freeze-dried siRNA/PEI powder for inhalation with high aerosol performance and strong pulmonary gene silencing activity Intratracheal Administration of siRNA Dry Powder Targeting Vascular Endothelial Growth Factor Inhibits Lung Tumor Growth in Mice Establishment of an Evaluation Method for Gene Silencing by Serial Pulmonary Administration of siRNA and pDNA Powders: Naked siRNA Inhalation Powder Suppresses Luciferase Gene Expression in the Lung Gene silencing in a mouse lung metastasis model by an inhalable dry small interfering RNA powder prepared using the supercritical carbon dioxide technique Effective mRNA pulmonary delivery by dry powder formulation of PEGylated synthetic KL4 peptide Formulation of High-Performance Dry Powder Aerosols for Pulmonary Protein Delivery Non-invasive delivery strategies for biologics Pulmonary delivery of powders and solutions containing recombinant human granulocyte colony-stimulating factor (rhG-CSF) to the rabbit Spray dried powders and powder blends of recombinant human deoxyribonuclease (rhDNase) for aerosol delivery Solid state characterization of spray-dried powders of recombinant human deoxyribonuclease (rhDNase) Formulation and process development of (recombinant human) deoxyribonuclease I as a powder for inhalation Excipient-free pulmonary insulin dry powder: Pharmacokinetic and pharmacodynamics profiles in rats Preparation, characterization, and pharmacodynamics of insulin-loaded fumaryl diketopiperazine microparticle dry powder inhalation Design of spray dried insulin microparticles to bypass deposition in the extrathoracic region and maximize total lung dose Evaluation of inhaled recombinant human insulin dry powders: pharmacokinetics, pharmacodynamics and 14-day inhalation Protein inhalation powders: spray drying vs spray freeze drying The effect of operating and formulation variables on the morphology of spray-dried protein particles Effect of mannitol crystallization on the stability and aerosol performance of a spray-dried pharmaceutical protein, recombinant humanized anti-IgE monoclonal antibody Effect of spray drying and subsequent processing conditions on residual moisture content and physical/biochemical stability of protein inhalation powders The effect of formulation excipients on protein stability and aerosol performance of spray-dried powders of a recombinant humanized anti-IgE monoclonal antibody Spray-drying performance of a bench-top spray dryer for protein aerosol powder preparation The use of amino acids to prepare physically and conformationally stable spray-dried IgG with enhanced aerosol performance Amino acid-based stable adalimumab formulation in spray freeze-dried microparticles for pulmonary delivery Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G Stabilization of IgG1 in spray-dried powders for inhalation A comparative study on the physicochemical and biological stability of IgG1 and monoclonal antibodies during spray drying process How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions Application of disaccharides alone and in combination, for the improvement of stability and particle properties of spray-freeze dried IgG Particle engineering of materials for oral inhalation by dry powder inhalers. I-Particles of sugar excipients (trehalose and raffinose) for protein delivery Peroxide formation in polysorbate 80 and protein stability Application of cyclodextrins in antibody microparticles: potentials for antibody protection in spray drying Spray-Freeze Drying: a Suitable Method for Aerosol Delivery of Antibodies in the Presence of Trehalose and Cyclodextrins Investigation of the dynamic process during spray-drying to improve aerodynamic performance of inhalation particles Optimization and characterization of spraydried IgG formulations: a design of experiment approach Respiratory Administration of Infliximab Dry Powder for Local Suppression of Inflammation Crystal structure of Cas9 in complex with guide RNA and target DNA Amorphous powders for inhalation drug delivery Alternatives to Conventional Antibiotics in the Era of Antimicrobial Resistance Native CRISPR-Cas-Mediated Genome Editing Enables Dissecting and Sensitizing Clinical Multidrug-Resistant P. aeruginosa CRISPR-Based Antibacterials: Transforming Bacterial Defense into Offense Heuze-Vourc'h, Insights on animal models to investigate inhalation therapy: Relevance for biotherapeutics Drying of a plasmid containing formulation: chitosan as a protecting agent Immunoglobulin G particles manufacturing by spray drying process for pressurised metered dose inhaler formulations Acknowledgements H-K Chan is grateful to Mr. Richard Stenlake for the generous financial support of his research.