Replication-deficient adenovirus-based gene therapy vectors were the first vectors demonstrated to mediate effective, robust in vivo gene transfer. The ease of genome engineering, large carrying capacity, and methods for large-scale vector production made adenoviral vectors a primary focus in the early days of gene therapy. Many vector modifications such as capsid engineering and regulated and cell-specific transgene expression were first demonstrated in adenovirus (Ad) vectors. However, early human studies proved disappointing, with safety and efficacy issues arising from anti-vector innate and acquired immune responses. While many gene therapy researchers moved to other vectors, others recognized that the immune response and limited duration of transgene expression were useful in the correct context. The striking example of this was the use of several effective adenovirus vectors engineered as COVID-19 vaccines estimated to have been administered to 2 billion people. In addition to vaccines, current applications of Ad vectors relate to anti-cancer therapies, tissue remodeling, and gene editing.

APOE4 homozygotes with Alzheimer’s have faster rates of cognitive decline and become symptomatic approximately a decade earlier. APOE2 is a protective variant with reduced likelihood of developing AD and a slower rate of decline. LX1001 is an adeno-associated viral vector investigational gene therapy (AAVrh.10hAPOE2) delivering the APOE2 gene into the central nervous system of APOE4 homozygotes to convert the brain APOE4 homozygous genotype to an APOE2/E4. This is a Phase 1/2, dose escalation study (NCT03634007) evaluating the safety and tolerability of LX1001 in four ascending single-dose cohorts (C1-C4). LX1001 was administered into the cerebrospinal fluid (CSF) at the craniocervical junction. Enrollment criteria include APOE4 homozygotes, age ≥ 50, positive amyloid PET, CSF biomarkers consistent with AD, and mild cognitive impairment (MCI) to moderate dementia. Following the one-time dose, the CSF APOE2/E4 profile, fluid and imaging biomarkers were assessed at regular intervals over 12 months. Fifteen participants were dosed: 50% MCI, 14% mild and 36% moderate dementia, at baseline. Twelve months of data are available for C1-C3 and 6 months for C4. Treatment with LX1001 was generally safe and well-tolerated. No events of amyloid related imaging abnormalities were observed . Post-treatment, APOE2 was expressed in CSF in all participants in a dose dependent manner. Interim results showed stabilization in CSF Aβ42/40 and amyloid PET. There was a decrease in CSF t-tau, p-tau, and Tau PET. Full data results including 12-month data for C4 will be presented during the meeting . LX1001 is the first investigational gene therapy for APOE4 homozygotes. Data suggest LX1001 is generally safe and well tolerated with reduction in CSF tau biomarkers and Tau PET, measures that are highly correlated with cognitive decline.

Rationale: The pathogenesis of COPD, the 3rd leading cause of death in the US, starts in the small airway epithelium (SAE). While smoking cessation slows the smoking-related decline of lung function, it does so less in COPD smokers (COPD-S) than in healthy smokers (S) and the rate of decline never returns to normal. Based on the knowledge that the transcriptome of the SAE is dysregulated in S and COPD-S with hundreds of genes up-and down-dysregulated compared to nonsmokers (NS), we hypothesized that, while all smokers have smoking-induced dysregulation of the SAE transcriptome, the dysregulation and the effect of smoking cessation is different in S compared to COPD-S and that remnants of smoking continue to stress the epithelium long after smoking has discontinued, likely contributing to progressive lung function loss in ex-smokers with COPD despite smoking cessation. Methods: SAE cells collected from 10th to 12th order bronchi using fiberoptic bronchoscopy were obtained at 4 different time points over 1 year from 27 S, 23 COPD-S and 12 NS. HG-U133 Plus 2.0 microarrays were used to identify the genes consistently dysregulated in response to smoking and COPD over 1 year (based on repeated measures ANOVA, fold-change ≥1.5, Benjamini-Hochberg corrected p <0.05). A separate cohort of 10 S and 12 COPD-S that were sampled while smoking, and then repetitively over 1 year after quitting smoking (S-Q and COPD-Q, respectively) was used to assess the effect of smoking cessation on the dysregulated SAE transcriptome. Repetitive sampling was performed at baseline, 3, 6 and 12 months for all subjects (total n=313 samples). Results: There were n=489 differentially expressed genes in COPD-S compared to NS and n=453 genes in S compared to NS. Of those, 354 genes (72%) were commonly dysregulated in S and COPD-S and n=135 genes (28%) uniquely dysregulated in COPD-S. Interestingly, following smoking cessation for 12 months, 7 genes (2%) remained dysregulated in S-Q, but in COPD-Q, 34 genes (8%) remained dysregulated. The non-reversible genes in COPD-Q included several COPD-related genes: SLIT2, MLKL, CYP1B1 and CH25H, in addition to the lung cancer-related genes PTPRH and ATP13A4-AS1. Conclusions: The SAE transcriptome is significantly and differentially dysregulated by smoking and by COPD. While smoking cessation reverses most of the effects of smoking, some COPD-dysregulated genes remain abnormal and may be responsible for continuous deterioration of lung health. These genes may be useful for gene targeting in drug therapy for COPD.

Rationale: Intensive care units (ICU) patients are highly vulnerable to inaccurate drug dosing. Pharmacogenomics (PGx) characterizes the influence of inherited genetic variation on drug metabolism, playing an important role in the consequences of a given drug dose. Objectives: To assess the genetic-based risk of inaccurate drug dosing in the ICU. Methods: We carried out whole genome sequencing (WGS) of 210 Qataris in ICU care at Hamad Medical Corporation (HMC), Doha, Qatar and assessed the WGS for predicted deleterious variants of genes that metabolize 30 drugs commonly prescribed in the ICU. Measurements and Main Results: Analysis of 210 Qatari ICU WGS identified 329 variants predicted deleterious associated with 85 genes known to affect metabolism of the 30 ICU drugs. Of the ICU patients that received the 5 most commonly prescribed drugs (warfarin, phenytoin, midazolam, vancomycin, levetiracetam), 93% had deleterious metabolism-related variants. Most (91%) patients carried at least one variant in a gene that that had the potential to affect the metabolism or activity of at least 1 drug that the patient received. Most patients had greater than or equal to 14 deleterious variants of genes that affect the metabolism of administered drugs. Comparison of the deleterious variants related to metabolism of ICU drugs with African/African American and European populations revealed significant population specificity in ICU related PGx variants. Conclusions: Together, these data suggest that population specific, pharmacogenomics based on the individual's genome likely plays a significant role in effective, safe dosing in the ICU setting. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement : These studies were supported, in part, by Qatar National Research Fund PPM 03-0314-190024 and Department of Genetic Medicine, Weill Cornell Medicine. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of Weill Cornell Medicine, Qatar gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the author

Rationale: Transposable elements (TE) are mobile sequences in the human genome that change location, have variable expression and function as cis-regulating elements that modify the expression of the transcriptome. Based on the knowledge that the small airway epithelium (SAE) is the 1st site of COPD-related early pathology caused by cigarette smoking, we hypothesized that: (1) smoking alters the expression of TE in the SAE; and (2) some smoker dysregulated SAE TE are associated with dysregulation of the SAE transcriptome. Methods: Illumina HiSeq 4000 RNAseq analysis was used to assess the TE and the expressed transcriptome of the SAE (10th to 12th order bronchi), collected by bronchoscopy and brushing, of cigarette smokers (n=20) and nonsmokers (n=26), all with normal lung function. Bioinformatics tools Telescope and Stellarscope were used for RNAseq and single-cell TE analysis of n=3 smokers and n=3 nonsmokers, respectively. TE expression with ≥5 counts in ≥3 samples was compared using DESeq2 among cigarette smokers and nonsmokers. Genes with Benjamini-Hochberg adjusted p value <0.05 and fold-change ≥1.5 were considered differentially expressed. Identification of the link between the differentially expressed TE and the SAE transcriptome was based on the location of TE, the directionality of regulation and fold-change in expression between smokers and nonsmokers. Results: Compared to nonsmokers, there were 155 downregulated and 205 upregulated differentially expressed TE in the SAE of cigarette smokers. Single cell RNAseq showed that intermediate and differentiated club, mucus and ciliated cells had the highest number of dysregulated TE in smokers. The impact of the differentially expressed TE on the expression of the closest transcriptome genes in smokers vs nonsmokers identified 29 downregulated TE associated with 23 downregulated transcriptome genes and 42 upregulated TE linked with 35 upregulated transcriptome genes. Importantly, many of the TE associated dysregulated genes play a role in lung carcinogenesis, including, CYP1B1, CYP1B1-AS1, EPHB1, ADH7, EGF, GCLC, MEI, AKR1B10, AKR1C2, MUCL1, AR, CCL17 and HBB. Other smoking dysregulated TE linked to dysregulated transcriptome genes are implicated in the pathogenesis of lung fibrosis, including ACSS3 and CCL17. Conclusions: Smoking-induced dysregulation of TE in the SAE of cigarette smokers in intermediate and differentiated cells is associated with dysregulation of genes in the SAE transcriptome, including genes linked to lung cancer and lung fibrosis. Understanding the impact of smoking on TE-mediated gene regulation may lead to the identification of new therapeutic targets for smoking-related lung diseases.

CLN2 disease (late infantile neuronal ceroid lipofuscinosis) is an autosomal recessive, neurodegenerative lysosomal storage disease that results from loss of function mutations in the CLN2 gene, which encodes tripeptidyl peptidase 1. It affects the central nervous system (CNS) with progressive neurodegeneration and early death, typically at ages from 8 to 12 years. Twenty years ago, our phase I clinical trial treated subjects with CLN2 disease by a catheter-based CNS administration of an adeno-associated virus vector serotype 2 (AAV2) expressing the CLN2 gene. Here we present an analysis of the survival of the 10 treated children and find 2 distinct survival groups with a wide disparity in survival. Group 1 (n = 7) had the typical mean survival of 8.8 ± 0.5 years of age, 3.8 ± 0.6 years post-therapy. Group 2 (n = 3) had a markedly longer mean survival of 23.4 ± 2.4 years of age and 14.9 ± 2.8 years post-therapy (p < 0.00002, survival of group 1 vs. group 2). Long survivors (group 2) at the time of treatment were older (group 1: 5.0 ± 0.6 years; group 2: 8.5 ± 0.9 years; p < 0.02); had similar disease severity (Hamburg clinical score group 1: 4.7 ± 0.5, group 2: 3 ± 0.0, p > 0.05); and had larger CNS ventricular volume (81.1 ± 22.2 cm3 vs. 27.3 ± 7.2 cm3 for group 1; p < 0.02). While the genotype of 3, group 2 subjects, had one allele (509-1G>C) identical to that of three in group 1, the second allele was different. This was unlikely to explain the survival difference, as alleles for both groups were equally predicted deleterious by the Combined Annotation-Dependent Depletion score: 34.9 ± 0.7 and 32.8 ± 0.3 for groups 1 and 2, respectively (a score of >20 is considered deleterious). This represents one of the longest survival studies (up to 20 years) of AAV-treated individuals with hereditary disorders and demonstrates variability of therapeutic efficacy where the genotype on its own has no apparent survival advantage. Protocol registration numbers for the original study: NCT00151216 and NCT00151268; www.clinicaltrials.gov.

BACKGROUND:APOE4 homozygotes with Alzheimer's have faster rates of cognitive decline and become symptomatic approximately a decade earlier. APOE2 is a protective variant with reduced likelihood of developing AD and a slower rate of decline. LX1001 is an adeno-associated viral vector investigational gene therapy (AAVrh.10hAPOE2) delivering the APOE2 gene into the central nervous system of APOE4 homozygotes to convert the brain APOE4 homozygous genotype to an APOE2/E4. METHOD:This is a Phase 1/2, dose escalation study (NCT03634007) evaluating the safety and tolerability of LX1001 in four ascending single-dose cohorts (C1-C4). LX1001 was administered into the cerebrospinal fluid (CSF) at the craniocervical junction. Enrollment criteria include APOE4 homozygotes, age ≥ 50, positive amyloid PET, CSF biomarkers consistent with AD, and mild cognitive impairment (MCI) to moderate dementia. Following the one-time dose, the CSF APOE2/E4 profile, fluid and imaging biomarkers were assessed at regular intervals over 12 months. RESULT:Fifteen participants were dosed: 50% MCI, 14% mild and 36% moderate dementia, at baseline. Twelve months of data are available for C1-C3 and 6 months for C4. Treatment with LX1001 was generally safe and well-tolerated. No events of amyloid related imaging abnormalities were observed. Post-treatment, APOE2 was expressed in CSF in all participants in a dose dependent manner. Interim results showed stabilization in CSF Aβ42/40 and amyloid PET. There was a decrease in CSF t-tau, p-tau, and Tau PET. Full data results including 12-month data for C4 will be presented during the meeting. CONCLUSION:LX1001 is the first investigational gene therapy for APOE4 homozygotes. Data suggest LX1001 is generally safe and well tolerated with reduction in CSF tau biomarkers and Tau PET, measures that are highly correlated with cognitive decline.

Mycobacterium tuberculosis (Mtb) must withstand physical and chemical stresses during airborne transmission, including during the desiccation of aerosols small enough to reach pulmonary alveoli in a new host. There, Mtb encounters an antimicrobial pulmonary alveolar lining fluid (ALF) before it is engulfed by macrophages. To study the genes involved in Mtb’s ability to survive the transition from desiccated droplet to pulmonary alveolus in an in vitro model, we formulated a model alveolar lining fluid (MALF) that mimics the composition of ALF as inferred from human bronchoalveolar lavage fluid (BALF). We compared the transcriptome of log-phase Mtb in MALF to the transcriptome of Mtb in BALF as BALF from the lungs of healthy adults was reconstituted to compensate for the dilution of ALF by lavage (rcBALF). Mtb from log-phase culture in a standard laboratory medium survived quantitatively in MALF and rcBALF for at least 24 hours. In contrast, Mtb that had passed through earlier stages of transmission began to succumb after 3 hours in MALF, past the time when particles have been observed to be phagocytized by alveolar macrophages. Screening of a genome-wide CRISPRi library of Mtb identified 35 genes as uniquely required by Mtb to survive the transition from desiccated microdroplet into rehydration in MALF. Thirty-one of these genes are non-essential under conventional laboratory conditions and seven have unknown functions. Thirteen of the 35 genes were additionally required for Mtb to survive in macrophage-like cells cultured at the air-liquid interface with pulmonary epithelial cells. This study nominates additional members of the transmission survival genome of Mtb, illustrates that different genes may contribute to the survival of Mtb at different stages of transmission, and suggests that modeled transmission can shed light on the functions of Mtb genes whose contributions have been unknown.

Gene therapy to treat hereditary disorders conventionally delivers the normal allele to compensate for loss-of-function mutations. More effective gene therapy may be achieved using a gain-of-function variant. We tested the hypothesis that AAVrh.10-mediated CNS delivery of the human APOE2 allele with the Christchurch mutation (R136S) (E2Ch) will provide superior protection against APOE4-associated Alzheimer's disease (AD) in mice compared to the unmodified APOE2 allele (E2). The vectors were assessed in two mouse strains with humanized APOE4: APP.PSEN1/TRE4 "amyloid mice" and P301S/TRE4, "tau mice." Both the E2Ch and E2 vectors prevented Aβ42 and Aβ40 accumulation and decreased β-amyloid aggregates in amyloid mice, but only the E2Ch vector suppressed tau tangles in tau mice. Microglial activation and reactive astrocytes were significantly suppressed by both vectors in amyloid mice but only the E2Ch vector mediated significant suppression of Iba1 and glial fibrillary acidic protein (GFAP) in tau mice. In four behavioral assays, the E2 and E2Ch vectors had similar benefits in amyloid mice, but E2Ch outperformed E2 in tau mice. In summary, while E2 is effective in suppressing amyloid pathology, the novel E2 variant E2Ch more effectively treats both the amyloid and tau pathology of murine AD in APOE4 background, supporting the development of AAVrh.10APOE2Ch as a therapy for APOE4-associated AD.

XC001 is a novel adenoviral-5 vector designed to express multiple isoforms of vascular endothelial growth factor to more safely and potently induce angiogenesis. The EXACT trial (NCT04125732) assessed the safety and preliminary efficacy of XC001 in patients with "no option" refractory angina (NORA). Single-arm, multicenter, open-label trial of 32 patients with NORA received a single treatment of XC001 (1×1011 viral particles) via transepicardial delivery. There were no SAEs attributed to study drug. Twenty expected SAEs in 13 patients were related to the surgical procedure. Total exercise duration (TED) increased from a mean±SD of 359.9±105.55 seconds at baseline to 448.2±168.45 (3 months), 449.2±175.9 (6 months), and 477.6±174.7 (12 months) (+88.3 [95% CI 37.1–139.5] and +84.5 [34.1–134.9], and +115.5 seconds [59.1-171.9] at 3, 6, and 12 months) (Fig. 1). Total myocardial perfusion deficit on positron emission tomography imaging decreased by 10.2% (95% CI -3.1–23.5%), 14.3% (2.8–25.7%), and 10.2% (-0.8-21.2%). Time to onset of ST depression during exercise tolerance testing increased by 105.2 (95% CI -27.9–238.3), 113.6 (28.8–198.4), and 103.1 seconds (26.7–179.5). Angina frequency decreased by -7.7 (95% CI 4.1–11.3), -6.6 (3.5–9.7), and -8.8 episodes (4.6–13.0). Angina class improved in 81% of participants at 6 months. XC001 administered via transepicardial delivery is safe and generally well-tolerated in NORA. Exploratory improvements in exercise duration, ischemic burden, and angina frequency and severity support a biologic effect sustained to 12 months, warranting further investigation.

The homozygous Apolipoprotein E (APOE4) genotype is the major risk factor for the development of early Alzheimer's disease. Genome engineering studies in mouse models of human APOE4-dependent pathology have established that reduction of APOE4 expression can rescue the phenotype. We hypothesized that APOE4 could be suppressed in the CNS of APOE4 homozygotes using adeno-associated virus (AAV) expression of microRNAs (miRNA) designed to hybridize to APOE mRNA. We screened nine different miRNAs targeting APOE following transfection in HEK293T and Huh7 cells. Optimal APOE suppression was obtained with mir2A (targeting coding region nt330-351) and mirN4 (3' untranslated region nt1142-1162). miRNA expression cassettes were designed with two copies of each of these two miRNAs co-expressed with a mCherry transgene. To optimize delivery of these miRNAs, an engineered AAVrh.10 variant was identified from a screen of multiple peptide insertions into capsid loop IV and substitutions in loop VIII. This led to identifying the AAV.S2 capsid with enhanced transduction of both neurons and glia and enhanced distribution in the brain. The engineered capsid was used to deliver the APOE miRNA suppression cassette to the hippocampus of TRE4 mice (human APOE4 knock-in replacement of the murine apoE locus). Two weeks after intra-hippocampus administration, regional expression of miRNA at the injection site was quantified at the mRNA level relative to an endogenous reference. The AAV.S2 capsid provided 2.31 ± 0.37-fold higher expression of miRNA over that provided by AAVrh.10 (p < 0.05). In the targeted region, a single intra-hippocampus AAV.S2 administration suppressed hippocampal APOE4 mRNA levels by 76.5 ± 3.9% compared with 41.3 ± 3.3% with the same cassette delivered by the wildtype AAVrh.10 capsid (p < 0.0001). We conclude that an expression cassette with two different miRNAs targeting APOE4 delivered by the AAV.S2 capsid will generate highly significant suppression of APOE4 in the CNS.

Friedreich's ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN. However, unlike endogenous hFXN, endogenous mFXN is further processed into N-terminally truncated, extra-mitochondrial mFXN forms of unknown function. This study assessed mature exogenous hFXN expression levels in the heart and liver of C57Bl/6 mice 7-10 months after intravenous administration of a recombinant adeno-associated virus encoding hFXN (AAVrh.10hFXN) and examined the potential for hFXN truncation in mice. AAVrh.10hFXN induced dose-dependent expression of hFXN in the heart and liver. Interestingly, hFXN was processed into truncated forms, but found at lower levels than mature hFXN. However, the truncations were at different positions than mFXN. AAVrh.10hFXN induced mature hFXN expression in mouse heart and liver at levels that approximated endogenous mFXN levels. These results suggest that AAVrh.10hFXN can likely induce expression of therapeutic levels of mature hFXN in mice.

BACKGROUND:Duchenne muscular dystrophy (DMD) is a rare, degenerative, recessive X-linked neuromuscular disease. Mutations in the gene encoding dystrophin lead to the absence of functional dystrophin protein. Individuals living with DMD exhibit progressive muscle weakness resulting in loss of ambulation and limb function, respiratory insufficiency, and cardiomyopathy, with multiorgan involvement. Adeno-associated virus vector-mediated gene therapy designed to enable production of functional dystrophin protein is a new therapeutic strategy. Delandistrogene moxeparvovec (Sarepta Therapeutics, Cambridge, MA) is indicated for treatment of ambulatory pediatric patients aged 4 through 5 years with DMD who have an indicated mutation in the DMD gene.OBJECTIVE:Evidence-based considerations for management of potential adverse events following gene therapy treatment for DMD are lacking in clinical literature. Our goal was to provide interdisciplinary consensus considerations for selected treatment-related adverse events (TRAEs) (vomiting, acute liver injury, myocarditis, and immune-mediated myositis) that may arise following gene therapy dosing with delandistrogene moxeparvovec.METHODS:An interdisciplinary panel of 12 specialists utilized a modified Delphi process to develop consensus considerations for the evaluation and management of TRAEs reported in delandistrogene moxeparvovec clinical studies. Panelists completed 2 Questionnaires prior to gathering for an in-person discussion. Consensus was defined as a majority (≥58% ; 7/12) of panelists either agreeing or disagreeing.RESULTS:Panelists agreed that the choice of baseline assessments should be informed by individual clinical indications, the treating provider's judgment, and prescribing information. Corticosteroid dosing for treatment of TRAEs should be optimized by considering individual risk versus benefit for each indication. In all cases involving patients with a confirmed TRAE, consultations with appropriate specialists were suggested.CONCLUSIONS:The Delphi Panel established consensus considerations for the evaluation and management of potential TRAEs for patients receiving delandistrogene moxeparvovec, including vomiting, acute liver injury, myocarditis, and immune-mediated myositis.