Brainmarker-I Differentially Predicts Remission to Various Attention-Deficit/Hyperactivity Disorder Treatments: A Discovery, Transfer, and Blinded Validation Study.

BACKGROUND: Attention-deficit/hyperactivity disorder is characterized by neurobiological heterogeneity, possibly explaining why not all patients benefit from a given treatment. As a means to select the right treatment (stratification), biomarkers may aid in personalizing treatment prescription, thereby increasing remission rates. METHODS: The biomarker in this study was developed in a heterogeneous clinical sample (N = 4249) and first applied to two large transfer datasets, a priori stratifying young males (<18 years) with a higher individual alpha peak frequency (iAPF) to methylphenidate (N = 336) and those with a lower iAPF to multimodal neurofeedback complemented with sleep coaching (N = 136). Blinded, out-of-sample validations were conducted in two independent samples. In addition, the association between iAPF and response to guanfacine and atomoxetine was explored. RESULTS: Retrospective stratification in the transfer datasets resulted in a predicted gain in normalized remission of 17% to 30%. Blinded out-of-sample validations for methylphenidate (n = 41) and multimodal neurofeedback (n = 71) corroborated these findings, yielding a predicted gain in stratified normalized remission of 36% and 29%, respectively. CONCLUSIONS: This study introduces a clinically interpretable and actionable biomarker based on the iAPF assessed during resting-state electroencephalography. Our findings suggest that acknowledging neurobiological heterogeneity can inform stratification of patients to their individual best treatment and enhance remission rates.

Binding of adenovirus species C hexon to prothrombin and the influence of hexon on vector properties in vitro and in vivo.

The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57 and 89. It is known that coagulation factors bind to Ad5 hexon and play a key role in the liver tropism of Ad5 vectors, but it is unclear how coagulation factors affect vectors derived from other species C Ads. We evaluated species C Ad vectors both in vitro and following intravenous injection in mice. To assess the impact of hexon differences, we constructed chimeric Ad5 vectors that contain the hexon hypervariable regions from other species C types, including vectors with hexon mutations that decreased coagulation factor binding. After intravenous injection into mice, vectors with Ad5 or Ad6 hexon had strong liver tropism, while vectors with chimeric hexon from other Ad types had weaker liver tropism due to inhibition by natural antibodies and complement. In addition, we discovered a novel ability of hexon to bind prothrombin, which is the most abundant coagulation factor in blood, and we found striking differences in the affinity of Ads for human, mouse and bovine coagulation factors. When compared to Ad5, vectors with non-Ad5 species C hexons had considerably higher affinity for both human and mouse prothrombin. Most of the vectors tested were strongly dependent on coagulation factors for liver transduction, but vectors with chimeric Ad6 hexon showed much less dependence on coagulation factors than other vectors. We found that in vitro neutralization experiments with mouse serum predicted in vivo behavior of Ad5 vectors, but in vitro experiments did not predict the in vivo behavior of vectors based on other Ad types. In sum, hexons from different human Ad species C viruses confer diverse properties on vectors, including differing abilities to target the liver.

Disruption of MeCP2-TCF20 complex underlies distinct neurodevelopmental disorders.

MeCP2 is associated with Rett syndrome (RTT), MECP2 duplication syndrome, and a number of conditions with isolated features of these diseases, including autism, intellectual disability, and motor dysfunction. MeCP2 is known to broadly bind methylated DNA, but the precise molecular mechanism driving disease pathogenesis remains to be determined. Using proximity-dependent biotinylation (BioID), we identified a transcription factor 20 (TCF20) complex that interacts with MeCP2 at the chromatin interface. Importantly, RTT-causing mutations in MECP2 disrupt this interaction. TCF20 and MeCP2 are highly coexpressed in neurons and coregulate the expression of key neuronal genes. Reducing Tcf20 partially rescued the behavioral deficits caused by MECP2 overexpression, demonstrating a functional relationship between MeCP2 and TCF20 in MECP2 duplication syndrome pathogenesis. We identified a patient exhibiting RTT-like neurological features with a missense mutation in the PHF14 subunit of the TCF20 complex that abolishes the MeCP2-PHF14-TCF20 interaction. Our data demonstrate the critical role of the MeCP2-TCF20 complex for brain function.

Intrinsic Functional Connectomes Characterize Neuroticism in Major Depressive Disorder and Predict Antidepressant Treatment Outcomes.

BACKGROUND: Antidepressant efficacy in people with major depressive disorder remains modest, yet identifying treatment-predictive neurobiological markers may improve outcomes. While disruptions in functional connectivity within and between large-scale brain networks predict poorer treatment outcome, it is unclear whether higher trait neuroticism, which has been associated with generally poorer outcomes, contributes to these disruptions and to antidepressant-specific treatment outcomes. Here, we used whole-brain functional connectivity analysis to identify a neural connectomic signature of neuroticism and tested whether this signature predicted antidepressant treatment outcome. METHODS: Participants were 226 adults with major depressive disorder and 68 healthy control subjects who underwent functional magnetic resonance imaging and were assessed on clinical features at baseline. Participants with major depressive disorder were then randomized to 1 of 3 commonly prescribed antidepressants and after 8 weeks completed a second functional magnetic resonance imaging and were reassessed for depressive symptom remission/response. Baseline intrinsic functional connectivity between each pair of 436 brain regions was analyzed using network-based statistics to identify connectomic features associated with neuroticism. Features were then assessed on their ability to predict treatment outcome and whether they changed after 8 weeks of treatment. RESULTS: Higher baseline neuroticism was associated with greater connectivity within and between the salience, executive control, and somatomotor brain networks. Greater connectivity across these networks predicted poorer treatment outcome that was not mediated by baseline neuroticism, and connectivity strength decreased after antidepressant treatment. CONCLUSIONS: Our findings demonstrate that neuroticism is associated with organization of intrinsic neural networks that predict treatment outcome, elucidating its biological underpinnings and opportunity for better treatment personalization.

Antidepressant side effects and their impact on treatment outcome in people with major depressive disorder: an iSPOT-D report.

Side effects to antidepressant medications are common and can impact the prognosis of successful treatment outcome in people with major depressive disorder (MDD). However, few studies have investigated the severity of side effects over the course of treatment and their association with treatment outcome. Here we assessed the severity of side effects and the impact of treatment type and anxiety symptoms over the course of treatment, as well as whether side effects were associated with treatment outcome. Participants were N = 1008 adults with a current diagnosis of single-episode or recurrent, nonpsychotic MDD. Participants were randomised to receive escitalopram, sertraline, or venlafaxine-extended release with equal probability and reassessed at 8 weeks regarding Hamilton Rating Scale Depression (HRSD17) and Quick Inventory of Depressive Symptomatology (QIDS-SR16) remission and response. Severity of side effects were assessed using the Frequency, Intensity, and Burden of Side Effects Rating (FIBSER) scale and assessed at day 4 and weeks 2, 4, 6, and 8. Frequency, intensity, and burden of side effects were greatest at week 2, then only frequency and intensity of side effects gradually decreased up to week 6. Treatment type and anxiety symptoms did not impact the severity of side effects. A greater burden-but not frequency or intensity-of side effects was associated with poorer treatment outcome and as early as 4 days post-treatment. Together, this work provides an informative mapping of the progression of side effects throughout the treatment course and their association with treatment outcome. Importantly, the burden of side effects that are present as early as 4 days post-treatment predicts poorer treatment outcome and should be monitored closely. iSPOT-D: Registry name: ClinicalTrials.gov. Registration number: NCT00693849.

Nitric oxide modulates bone anabolism through regulation of osteoblast glycolysis and differentiation.

Previous studies have shown that nitric oxide (NO) supplements may prevent bone loss and fractures in preclinical models of estrogen deficiency. However, the mechanisms by which NO modulates bone anabolism remain largely unclear. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing arginine, the sole precursor for nitric oxide synthase-dependent (NOS-dependent) NO synthesis. Moreover, ASL is also required for channeling extracellular arginine to NOS for NO production. ASL deficiency (ASLD) is thus a model to study cell-autonomous, NOS-dependent NO deficiency. Here, we report that loss of ASL led to decreased NO production and impairment of osteoblast differentiation. Mechanistically, the bone phenotype was at least in part driven by the loss of NO-mediated activation of the glycolysis pathway in osteoblasts that led to decreased osteoblast differentiation and function. Heterozygous deletion of caveolin 1, a negative regulator of NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass in a hypomorphic mouse model of ASLD. The translational significance of these preclinical studies was further reiterated by studies conducted in induced pluripotent stem cells from an individual with ASLD. Taken together, our findings suggest that ASLD is a unique genetic model for studying NO-dependent osteoblast function and that the NO/glycolysis pathway may be a new target to modulate bone anabolism.

Dynamic Dossier in the Cloud: A Sociotechnical Architecture for a Real-Time and Metrics-Based Data Tracking System with Gene and Cell Therapies as a Case Study.

BACKGROUND: Data sharing among stakeholders in the development, access, and use of drug therapies is critical but the current system and process are inefficient. METHODS: We take a Systems Engineering approach with a realistic use case to propose a scalable design for multi-stakeholder data sharing. RESULTS: We make three major contributions to the drug development and healthcare communities: first, a methodology for developing a multi-stakeholder data sharing system, with its focus on high-level requirements that influence the design of the system architecture and technology choice; second, the development of a realistic use case for long-term patient and therapy data tracking and sharing in the use of potentially curative and durable gene and cell therapies. Further, a bridge for the 'awareness gap' was found between the payer (Payer is organization which takes care of financial and operational aspects (which include insurance plans, provider network) of providing health care to US citizens. Or refer to health care insurers.) and the regulator communities by illustrating the common data tracking needs, which highlights the need for coordinated data activities; and third, a proposed system architecture for scalable, multi-stakeholder data sharing. Next steps are briefly discussed. CONCLUSION: We present a system design for multiple stakeholders such as the payer, the regulator, the developer (drug manufacturer), and the healthcare provider to share data for their decision-making. The stakeholder community would benefit from collaboratively moving the system development proposal forward for efficient and cost-effective data sharing.

A Single "All-in-One" Helper-Dependent Adenovirus to Deliver Donor DNA and CRISPR/Cas9 for Efficient Homology-Directed Repair.

In this study, we developed a single helper-dependent adenovirus (HDAd) to deliver all of the components (donor DNA, CRISPR-associated protein 9 [Cas9], and guide RNA [gRNA]) needed to achieve high-efficiency gene targeting and homology-directed repair in transduced cells. We show that these "all-in-one" HDAds are up to 117-fold more efficient at gene targeting than donor HDAds that do not express CRISPR/Cas9 in human induced pluripotent stem cells (iPSCs). The vast majority (>90%) of targeted recombinants had only one allele targeted, and this was accompanied by high-frequency indel formation in the non-targeted allele at the site of Cas9 cleavage. These indels varied in size and nature, and included large deletions of ∼8 kb. The remaining minority of recombinants had both alleles targeted (so-called bi-allelic targeting). These all-in-one HDAds represent an important platform for accomplishing and expanding the utility of homology-directed repair, especially for difficult-to-transfect cells and for in vivo applications.

Mapping axon initial segment structure and function by multiplexed proximity biotinylation.

Axon initial segments (AISs) generate action potentials and regulate the polarized distribution of proteins, lipids, and organelles in neurons. While the mechanisms of AIS Na+ and K+ channel clustering are understood, the molecular mechanisms that stabilize the AIS and control neuronal polarity remain obscure. Here, we use proximity biotinylation and mass spectrometry to identify the AIS proteome. We target the biotin-ligase BirA* to the AIS by generating fusion proteins of BirA* with NF186, Ndel1, and Trim46; these chimeras map the molecular organization of AIS intracellular membrane, cytosolic, and microtubule compartments. Our experiments reveal a diverse set of biotinylated proteins not previously reported at the AIS. We show many are located at the AIS, interact with known AIS proteins, and their loss disrupts AIS structure and function. Our results provide conceptual insights and a resource for AIS molecular organization, the mechanisms of AIS stability, and polarized trafficking in neurons.

Dimensions of anxiety in Major depressive disorder and their use in predicting antidepressant treatment outcome: an iSPOT-D report.

BACKGROUND: Major depressive disorder (MDD) commonly co-occurs with clinically significant levels of anxiety. However, anxiety symptoms are varied and have been inconsistently associated with clinical, functional, and antidepressant treatment outcomes. We aimed to identify and characterise dimensions of anxiety in people with MDD and their use in predicting antidepressant treatment outcome. METHOD: 1008 adults with a current diagnosis of single-episode or recurrent, nonpsychotic, MDD were assessed at baseline on clinical features and cognitive/physiological functioning. Participants were then randomised to one of three commonly prescribed antidepressants and reassessed at 8 weeks regarding symptom change, as well as remission and response, on the 17-item Hamilton Rating Scale Depression (HRSD17) and the 16-item Quick Inventory of Depressive Symptomatology (QIDS-SR16). Exploratory factor analysis was used on items from scales assessing anxiety symptoms, and resulting factors were assessed against clinical features and cognitive/physiological functioning. Factors were also assessed on their ability to predict treatment outcome. RESULTS: Three factors emerged relating to stress, cognitive anxiety, and somatic anxiety. All factors showed high internal consistency, minimal cross-loadings, and unique clinical and functional profiles. Furthermore, only higher somatic anxiety was associated with poorer QIDS-SR16 remission, even after adjusting for covariates and multiple comparisons. CONCLUSIONS: Anxiety symptoms in people with MDD can be separated onto distinct factors that differentially respond to treatment outcome. Furthermore, these factors do not align with subscales of established measures of anxiety. Future research should consider cognitive and somatic symptoms of anxiety separately when assessing anxiety in MDD and their use in predicting treatment outcome.

Annual variation in attentional response after methylphenidate treatment.

Prevalence rates of attention-deficit/hyperactivity disorder (ADHD) differ with geographical areas varying in sunlight intensity. Sun- or daylight reaching the retina establishes entrainment of the circadian clock to daylight. Changes herein, hence, alterations in clock alignment, could be reflected indirectly in inattention via sleep duration. We here studied (1) annual variation in inattention at treatment initiation; (2) annual variation in response to ADHD treatment [methylphenidate (MPH)] by day of treatment initiation; and (3) dose dependence. We predicted least baseline inattention during a period of high sunlight intensity implying more room for improvement (i.e., a better treatment response) when sunlight intensity is low. These hypotheses were not confirmed. High-dose treated patients, however, had significantly better attention after treatment than low-dosed treated patients, only when treated in the period from winter to summer solstice. Change in solar irradiance (SI) during low-dosed treatment period was negatively related to attentional improvement. The above described findings were primarily found in inattention ratings and replicated in omission errors on a continuous performance task. Daylight and inattention have been proposed to be related via mediation of the circadian system. One mechanism of MPH may be to enhance sensitivity to the diurnal entrainment to sunlight and the question can be raised whether appropriate lighting could potentiate the effects of stimulants.

Verbal memory predicts treatment outcome in syndromal anxious depression: An iSPOT-D report.

BACKGROUND: Major Depressive Disorder (MDD), anxiety disorders, and high levels of anxious symptoms are associated with impaired cognitive functioning. However, little is known of how cognitive functioning is impaired in people with anxious depression. Here, we compared cognitive functioning between people with anxious depression, non-anxious depression, and healthy controls. We also tested whether anxious depression moderated the relationship between cognitive functioning and treatment outcome. METHODS: 1008 adults with MDD and 336 healthy controls completed IntegNeuro: a computerized cognitive functioning test battery. Participants were then randomised to one of three antidepressants and reassessed at 8 weeks using the 17-item Hamilton Depression Rating Scale (HRSD17) and the 16-Item Quick Inventory of Depressive Symptomatology-Self-Rated for remission and response. Syndromal anxious depression was defined as MDD with a comorbid anxiety disorder. HRSD anxious depression was defined as MDD with a comorbid HRSD17 anxiety/somatisation factor score ≥ 7. RESULTS: Syndromal anxious depression was associated with better psychomotor functioning and poorer working memory, cognitive flexibility and information processing speed compared to their non-anxious counterparts. HRSD anxious depression was associated with better psychomotor functioning compared to their non-anxious counterparts. Syndromal anxious depression moderated the relationship between verbal memory and treatment outcome. In people with syndromal anxious depression, poorer baseline verbal memory predicted poorer treatment outcome. LIMITATIONS: As DSM-IV criteria was used, the DSM-5 anxious distress specifier characterisation of anxious depression could not be assessed CONCLUSIONS: Syndromal anxious depression is characterised by impaired executive functions and moderates the relationship between verbal memory functioning and treatment outcome.

Probing the "Default Network Interference Hypothesis" With EEG: An RDoC Approach Focused on Attention.

Studies have shown that specific networks (default mode network [DMN] and task positive network [TPN]) activate in an anticorrelated manner when sustaining attention. Related EEG studies are scarce and often lack behavioral validation. We performed independent component analysis (ICA) across different frequencies (source-level), using eLORETA-ICA, to extract brain-network activity during resting-state and sustained attention. We applied ICA to the voxel domain, similar to functional magnetic resonance imaging methods of analyses. The obtained components were contrasted and correlated to attentional performance (omission errors) in a large sample of healthy subjects (N = 1397). We identified one component that robustly correlated with inattention and reflected an anticorrelation of delta activity in the anterior cingulate and precuneus, and delta and theta activity in the medial prefrontal cortex and with alpha and gamma activity in medial frontal regions. We then compared this component between optimal and suboptimal attentional performers. For the latter group, we observed a greater change in component loading between resting-state and sustained attention than for the optimal performers. Following the National Institute of Mental Health Research Domain Criteria (RDoC) approach, we prospectively replicated and validated these findings in subjects with attention deficit/hyperactivity disorder. Our results provide further support for the "default mode interference hypothesis."

Production of CRISPR/Cas9-Mediated Self-Cleaving Helper-Dependent Adenoviruses.

Prolonged expression of CRISPR/Cas9 raises concerns about off-target cleavage, cytotoxicity, and immune responses. To address these issues, we have developed a system to produce helper-dependent adenoviruses that express CRISPR/Cas9 to direct cleavage of the vectors' own genome after transduction of target cells. To prevent self-cleavage during vector production, it was necessary to downregulate Cas9 mRNA as well as inhibit Cas9 protein activity. Cas9 mRNA downregulation was achieved by inserting the target sequences for the helper-virus-encoded miRNA, mivaRNAI, and producer-cell-encoded miRNAs, hsa-miR183-5p, and hsa-miR218-5p, into the 3' UTR of the HDAd-encoded Cas9 expression cassette. Cas9 protein activity was inhibited by expressing anti-CRISPR proteins AcrIIA2 and AcrAII4 from both the producer cells and the helper virus. After purification, these helper-dependent adenoviruses will perform CRISPR/Cas9-mediated self-cleavage in the transduced target cells, thereby limiting the duration of Cas9 expression and thus represent an important platform for improving the safety of gene editing by CRISPR/Cas9.

Characterising anxiety in major depressive disorder and its use in predicting antidepressant treatment outcome: An iSPOT-D report.

OBJECTIVE: Major depressive disorder commonly co-occurs with one or more anxiety disorders or with clinically significant levels of anxiety symptoms. Although evidence suggests that anxious forms of depression are prognostic of poorer antidepressant outcomes, there is no clear definition of anxious depression, and inferences about clinical outcomes are thus limited. Our objective was to compare and evaluate definitions of anxious depression and anxiety-related scales according to clinical and antidepressant outcome criteria. METHOD: A total of 1008 adults with a current diagnosis of single-episode or recurrent, nonpsychotic, major depressive disorder were assessed at baseline on clinical features. Participants were then randomised to one of three antidepressants and reassessed at 8 weeks regarding remission and response of the 17-item Hamilton Rating Scale Depression (HRSD17) and the 16-item Quick Inventory of Depressive Symptomatology (QIDS-SR16). Anxious depression was defined as major depressive disorder with one or more anxiety disorders or major depressive disorder with a HRSD17 anxiety/somatisation factor score ⩾7. Anxiety-related scales included the HRSD17 anxiety/somatisation factor and the 42-item Depression Anxiety Stress Scales (DASS42) anxiety and stress subscales. RESULTS: Anxious depression definitions showed poor agreement (κ = 0.15) and the HRSD17 anxiety/somatisation factor was weakly correlated with both DASS42 anxiety (r = 0.24) and stress subscales (r = 0.20). Anxious depression definitions were also associated with few impairments on clinical features and did not predict poorer antidepressant treatment outcome. However, higher DASS42 anxiety predicted poorer HRSD17 and QIDS-SR16 remission, and item-level analysis found higher scores on items 9 (situational anxiety) and 23 (somatic anxiety) of the DASS42 predicted poorer treatment outcome, even after adjusting for covariates and multiple comparisons. CONCLUSION: Common definitions of anxious depression show poor agreement and do not predict poorer treatment outcome. Anxiety symptoms may be better characterised dimensionally using DASS42 when predicting treatment outcome.

Increased chronic stress predicts greater emotional negativity bias and poorer social skills but not cognitive functioning in healthy adults.

Background and Objectives: Chronically stressed individuals report deficits spanning cognitive and emotional functioning. However, limitations to clinical populations and measures of stress have impeded the generalisability and scope of results. This study investigated whether chronic stress predicted cognitive and emotional functioning, and whether these relationships differed between males and females, in a large representative sample of healthy participants. Design: Cross-sectional study. Method: 1883 healthy adults sampled from the Brain Resource International Database reported stress using the 21-item Depression Anxiety Stress Scales. Participants then completed a cognitive and emotional assessment battery (IntegNeuro), as well as questionnaires related to sleep, emotional functioning, and self-regulation. Results: In contrast to previously reported results, chronic stress did not predict cognitive functioning. However, higher stress predicted a greater negativity bias and poorer social skills, confirming previous research identifying these links. Conclusions: Cognitive deficits related to stress are absent in healthy participants when stress is measured using the 21-items Depression Anxiety Stress Scales. Identifying how chronic stress is associated with aspects of emotional functioning can lead to personalized interventions for individuals to better manage the negative outcomes resulting from stress.

Hepatic glutamine synthetase augmentation enhances ammonia detoxification.

The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-alpha-fetoprotein), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia.

A Signature of Attention-Elicited Electrocortical Activity Distinguishes Response From Non-Response to the Non-Stimulant Atomoxetine in Children and Adolescents With ADHD.

OBJECTIVE: Atomoxetine has several characteristics that make it an attractive alternative to stimulants for treating ADHD, but there are currently no tests identifying individuals for whom the medication should be a first-line option. METHOD: Within the ADHD Controlled Trial Investigation Of a Non-stimulant (ACTION) study, we examined neuro-cortical activity in 52 youth with ADHD. Baseline event-related potentials (ERP) were compared between those who subsequently responded to 6 weeks of atomoxetine versus those who did not. RESULTS: Responders were distinguished by significantly lower auditory oddball N2 amplitudes than both non-responders and typically developing controls, particularly in the right frontocentral region ( p = .002, Cohen's d = 1.1). Leave-one-out cross validation determined that N2 amplitude in this region was able to accurately predict non-responders with a specificity of 80.8%. There were no P3 differences between responders and non-responders. CONCLUSION: The N2 amplitude is a biomarker that may have utility in predicting response to atomoxetine for youth with ADHD.

Bi-allelic Homology-Directed Repair with Helper-Dependent Adenoviruses.

We describe a strategy to achieve footprintless bi-allelic homology-directed repair (HDR) using helper-dependent adenoviruses (HDAds). This approach utilizes two HDAds to deliver the donor DNA. These two HDAds are identical except for their selectable marker. One expresses the puromycin N-acetyltransferase-herpes simplex virus I thymidine kinase fusion gene (PACTk), while the other expresses the hygromycin phosphotransferase-herpes simplex virus I thymidine kinase fusion gene (HyTk). Therefore, puromycin and hygromycin double resistance can be used to select for targeted HDAd integration into both alleles. Subsequently, piggyBac-mediated excision of both PACTk and HyTk will confer resistance to gancyclovir, resulting in footprintless HDR at both alleles. However, gene-targeting frequency was not high enough to achieve simultaneous targeting at both alleles. Instead, sequential targeting, whereby the two alleles were targeted one at a time, was required in order to achieve bi-allelic HDR with HDAd.