Electroporation System Research Articles

Optimization of Gene Editing in Human Primary T Cells with Neon® Transfection System

Abstract The successful cases of autologous CAR-T cell therapy in leukemia have highlighted the promising future of cell therapy. However, most studies in this field have been using viruses to engineer T cells, which lead to safety concerns. On the other hand, there has been a growing interest in developing allogeneic immune cell therapies to tackle the challenges of production scale up. Hence, there is a need for a better delivery system to maximize gene editing efficiency in immune cells. Neon® Transfection System has been widely used in a variety of cell models and especially hard-to-transfect cells, such as primary cells and stem cells. Here we demonstrate how Neon® is fully capable of supporting all spectrums of research in cell therapy, including RNA delivery, DNA delivery, and CRISPR/Cas9 gene editing. Human primary T cells from four different donors were isolated from individual fresh Leukapheresis Pak, followed by activation/expansion in OpTmizer media with 2% human serum and CD3/CD28 Dynabeads. The optimal delivery time was between 3–5 days after activation. The transfection efficiency can be reached over 95% for RNA (EGFP mRNA) and 85% for DNA (pcDNAEF1a-emGFP) with high viability using certain Neon® programs. For CRISPR/Cas9 gene editing, more than 30% knock-in efficiency was observed while cleavage efficiency at HPRT locus can be reached more than 70%. The promising results of gene delivery and editing in primary T cells from Neon® could facilitate the progress of immunotherapy. Meanwhile, we have been dedicating in developing a next generation cGMP-certified large-scale electroporation system, which enable researchers to seamlessly scale-up from Neon®.

Voltage Charging Gold-Microtube Membranes for Electroporation

Electroporation of cell membrane occurs when sufficiently large electric field gradients (in the order of kV/cm) are applied to a cell medium. This is routinely accomplished in medicine and biology using voltage pulses above 1 kV in order to incorporate foreign molecules and genes into living cells.1 However, transient pore formation or reversible electroporation is a challenge with such large voltages because of Joule heating, pH changes and ion dissolution that lead to high cell mortality.2 Furthermore, special safety precautions are required. As such, the miniaturization of electroporation devices has been a research interest in order to reach similar electric field gradients with lower voltages.3 It was also shown that these microscopic devices result in higher electroporation yields and better cell viability, although often reducing the cell throughput. We have developed an electroporation system that consists on charging gold microtubes inside a filter membrane to electroporate Escherichia coli at low voltages (<5 V).4 Supporting our experimental results with theoretical simulations, we have shown that electric field gradients of 4 kV/cm, sufficiently large to reversibly porate the bacteria membrane, were formed inside the gold microtubes. Following the incorporation of exogenous probes inside E. coli with fluorescence microscopy and spectroscopy, we have determined that up to 40% of the bacteria were reversibly electroporated. This is more than an order of magnitude higher than the efficiency obtained with a commercial electroporation device. Finally, cell throughput of >30 million cells per minutes, higher than any previously reported device, were obtained. Gehl, J. Acta Physiol. Scand. 2003, 177, 437-447.Lee, W. G.; Demirci, U.; Khademhosseini, A. Integr. Biol. 2009, 1, 242-251.Geng, T.; Lu, C. Lab Chip 2013, 13, 3803-3821.Experton, J.; Wilson, A. G.; Martin C. R. Anal. Chem. [Article ASAP]. DOI: 10.1021/acs.analchem.6b03820. Published Online: Nov 17, 2016.

Pulsed Electric Fields Pretreatments for the Cooking of Foods

Development of the concept of electroporation opened new perspectives for promising applications in food technology. Treatment of foods with pulsed electric fields (PEFs) allows facilitation of different food transformation operations (extraction, expression, osmotic treatment, drying, and freezing) with minimal energy consumptions and better retention of flavor, color, and preservation of nutritional properties of foods. This work shortly reviews the effects of PEF on the biological cells and food products and gives the examples of PEF-assisted techniques. The PEF protocol, power consumption, and existing small- and large-scale electroporation systems are presented. Some examples of PEF-assisted processing of meat, fish, and fat frying are discussed. The main principles of PEF-assisted cooking and kitchen operations are also discussed. The variants of PEF-assisted non-thermal cooker and PEF/ohmic thermal cooker are presented. It is speculated that PEF allows more homogeneous treatment of foods as compared to the conventional methods of thermal cooking. The PEF-assisted cooking can be faster and more effective for nutrient retention and sensory qualities of foods. Moreover, the PEF treatment can be used for producing the types of the products of fresh/natural quality and new tastes. The recent examples of PEF-assisted processing of meat and fish, assistance of frying, and commercial-scale processing are also presented and discussed.

Microscale Symmetrical Electroporator Array as a Versatile Molecular Delivery System

Successful developments of new therapeutic strategies often rely on the ability to deliver exogenous molecules into cytosol. We have developed a versatile on-chip vortex-assisted electroporation system, engineered to conduct sequential intracellular delivery of multiple molecules into various cell types at low voltage in a dosage-controlled manner. Micro-patterned planar electrodes permit substantial reduction in operational voltages and seamless integration with an existing microfluidic technology. Equipped with real-time process visualization functionality, the system enables on-chip optimization of electroporation parameters for cells with varying properties. Moreover, the system’s dosage control and multi-molecular delivery capabilities facilitate intracellular delivery of various molecules as a single agent or in combination and its utility in biological research has been demonstrated by conducting RNA interference assays. We envision the system to be a powerful tool, aiding a wide range of applications, requiring single-cell level co-administrations of multiple molecules with controlled dosages.

Transfection of Jurkat T cells by droplet electroporation

A droplet electroporation system has been successfully demonstrated for transfection of Jurkat T cell with much higher transfection efficiency (66%) than that of conventional system (11.4%) with the advantages of superior cell viability, comparable throughput, and user-friendly interface. Because the productivity of the proposed system is much greater than previous microfluidic systems, flow cytometry is used for the analysis of transfection efficiency. The high transfection efficiency and cell viability of the droplet electroporation system is mainly attributed to small size which requires lower voltage and provides concentrated environment. The successful transfection of Jurkat cell using the droplet electroporation system broadens the applicability of the proposed technology to medical field. The implication of the present work and the future development direction for a fully automated cell engineering platform are discussed.

High-throughput Nuclear Delivery and Rapid Expression of DNA via Mechanical and Electrical Cell-Membrane Disruption.

Nuclear transfection of DNA into mammalian cells is challenging yet critical for many biological and medical studies. Here, by combining cell squeezing and electric-field-driven transport in a device that integrates microfluidic channels with constrictions and microelectrodes, we demonstrate nuclear delivery of plasmid DNA within 1 hour after treatment, the most rapid DNA expression in a high-throughput setting (up to millions of cells per minute per device). Passing cells at high speed through microfluidic constrictions smaller than the cell diameter mechanically disrupts the cell membrane, allowing a subsequent electric field to further disrupt the nuclear envelope and drive DNA molecules into the cytoplasm and nucleus. By tracking the localization of the ESCRT-III (endosomal sorting complexes required for transport) protein CHMP4B, we show that the integrity of the nuclear envelope is recovered within 15 minutes of treatment. We also provide insight into subcellular delivery by comparing the performance of the disruption-and-field-enhanced method with those of conventional chemical, electroporation, and manual-injection systems.

Biomimetic Lipid-Based Nanosystems for Enhanced Dermal Delivery of Drugs and Bioactive Agents.

Clinical utility of conventional oral therapies is limited by their inability to deliver therapeutic molecules at the local or targeted site, causing a variety of side effects. Transdermal delivery has made a significant contribution in the management of skin diseases with enhanced therapeutic activities over the past two decades. In the modern era, various biomimetic and biocompatible polymer-lipid hybrid systems have been used to augment the transdermal delivery of therapeutics such as dermal patches, topical gels, iontophoresis, electroporation, sonophoresis, thermal ablation, microneedles, cavitational ultrasound, and nano or microlipid vesicular systems. Nevertheless, the stratum corneum still represents the main barrier to the delivery of vesicles into the skin. Lipid based formulations applied to the skin are at the center of attention and are anticipated to be increasingly functional as the skin offers many advantages for the direction of such systems. Accordingly, this review provides an overview of the development of conventional to advanced biomimetic lipid vesicles for skin delivery of a variety of therapeutics, with special emphasis on recent developments in this field including the development of transferosomes, niosomes, aquasomes, cubosomes, and other new generation lipoidal carriers.

Electric field enhanced 3D scalable low-voltage nano-spike electroporation system

Electroporation (EP) is one of the most widely used methods for the introduction of bio-molecules into the cells due to its high efficiencies, simplicity, and safety. Previous macro- and micro-scale EP systems suffer from drawbacks such as costly and time-consuming fabrication processes, high voltage operation causes undesirable electrochemical reactions, low cell viabilities, and electrode degradation. In this paper, we presented a low-cost three-dimensional (3D) scalable nano-spike electroporation system for efficient molecules delivery with high cell viabilities at low applied voltages. Arrays of 3D Aluminum (Al) nano-spikes (NSPs) were fabricated through scalable, reproducible and cost effective electrochemical anodization and etching processes. Due to scalability of the fabrication process, 3D NSPs were fabricated on chips as well as at the wafer level for large scale processing. 3D nano-spike electroporation (NSP-EP) chips were capable of handling small cell populations (100–500) while NSP-EP wafers can handle large cell populations (104–105). Electroporation at low voltages is obtained due to electric field enhancement at high-aspect-ratio NSPs. With same electric field strength, high EP efficiencies ƞEP and cell viability фcell (>93±6%) were obtained at more than ten times lower voltages (2V) on NSP-EP chips as compared to planar electroporation (PEP) devices without NSPs. By optimizing electric pulse parameters and nano-spikes dimensions, NSP-EP chip performance was enhanced by minimizing undesirable electrochemical reactions and electrolysis that were observed on PEP devices due to high voltage operations.

CNTF protects neurons from hypoxic injury through the activation of STAT3pTyr705.

The aim of the present study was to investigate whether ciliary neurotrophic factor(CNTF) plays its neuroprotective role following hypoxic injury through the activation of signal transducer and activator of transcription3(STAT3) signaling. Firstly, to determine whether CNTF exerts its effects via STAT3 following hypoxic injury, cultured neurons from the cerebral cortex of mice were prepared and a neuronal model of hypoxia was then established. The neurons exposed to hypoxia were then pre-treated with CNTF and transfected with small interference RNA(siRNA) targeting STAT3(STAT3siRNA) using polybrene, or with STAT3Tyr705 mutant or STAT3Ser727 mutant using an electroporation system. The survival, proliferation and neurite outgrowth of the neurons subjected to different treatments were also determined. RT-qPCR and western blot analysis were employed to examine the expression levels of STAT3, p-STAT3Tyr705 and p-STAT3Ser727 following treatment with CNTF and other treatments. Our results revealed that treatment with CNTF: i)protected neurons from hypoxic injury by promoting survival and neurite growth; ii)induced a significant increase in the levels of STAT3, STAT3pTyr705 and the STAT3pTyr705/STAT3 ratio; it did not however, significantly affect the levels of STAT3pSer727 in the hypoxic cerebral cortex neurons. Transfection of the hypoxic neurons pre-treated with CNTF with STAT3siRNA or STAT3Tyr705 neutralized the protective effects exerted by CNTF. The findings of our study thus demonstrate that CNTF protects neurons from hypoxic injury through the activation of STAT3pTyr705.

276P - Electrochemotherapy for breast cancer - results from the INSPECT database

Cutaneous recurrence from breast cancer can pose a clinical challenge. It may be the only disease site, or be part of disseminated disease, and often profoundly impacts quality of life. Electrochemotherapy is a palliative treatment for cutaneous metastases. Using electric pulses to locally permeabilize tumor cells, bleomycin cytotoxicity is significantly increased. Collaborating in the International Network for sharing Practice on ElectroChemoTherapy (INSPECT), we consecutively and prospectively accrued data on patients treated with electrochemotherapy for skin metastases of breast cancer. Patients with cutaneous metastases were treated with electrochemotherapy at 10 European centers. Treatment data and results were entered into the INSPECT database. Patients were treated with either local injection (1000 IU/ml intratumoral injection) or systemic infusion (15.000 IU/m2) of bleomycin, and under either local or general anesthesia, depending on tumor size. Pulse sequences (8 pulses of 0.1 ms) were sequentially applied to the tumor area, using an electroporation system with needle or plate electrodes (IGEA, Italy). 104 patients were included. Median age was 65 years. Patients had previous received chemotherapy (87%), radiotherapy (83%), endocrine therapy (46%) and HER2 targeted therapy (20%). Primary location was the chest (87%), median diameter of the cutaneous metastases being 26 mm (range 1 mm to 550 mm). 75 patients were available for response evaluation after 2 months. Complete response was observed in 38 (51%) patients, partial response in 13 (17%), stable disease in 14 (19%), and progressive disease in 5 (9%), 3 patients were not evaluable. Common side effects were ulceration, long lasting hyperpigmentation, and slight increase in pain. No serious adverse events were observed. Electrochemotherapy showed high response rates, particularly in smaller metastases. Electrochemotherapy has high response rates seen after a single treatment, with few side effects and can be used as an adjunct to systemic therapies as well as a sole treatment. We therefore highly recommend to consider electrochemotherapy for patients with cutaneous metastases, where radiotherapy and surgery is not an option.

Paralleled comparison of vectors for the generation of CAR-T cells.

T-lymphocytes genetically engineered with the chimeric antigen receptor (CAR-T) have shown great therapeutic potential in cancer treatment. A variety of preclinical researches and clinical trials of CAR-T therapy have been carried out to lay the foundation for future clinical application. In these researches, several gene-transfer methods were used to deliver CARs or other genes into T-lymphocytes, equipping CAR-modified T cells with a property of recognizing and attacking antigen-expressing tumor cells in a major histocompatibility complex-independent manner. Here, we summarize the gene-transfer vectors commonly used in the generation of CAR-T cell, including retrovirus vectors, lentivirus vectors, the transposon/transposase system, the plasmid-based system, and the messenger RNA electroporation system. The following aspects were compared in parallel: efficiency of gene transfer, the integration methods in the modified T cells, foreground of scale-up production, and application and development in clinical trials. These aspects should be taken into account to generate the optimal CAR-gene vector that may be suitable for future clinical application.

Efficient transformation of Staphylococcus aureus using multi-pulse electroporation

A new multi-pulse electroporation system was evaluated to transform Staphylococcus aureus. Compared to the conventional electroporation system, it yielded high transformation efficiency to obtain more than 3.9×105S. aureus RN4220 transformed cells/1μg plasmid DNA using a single electroporation by manipulating the poring pulse and transfer pulse.

Flow-Through Electroporation of HL-60 White Blood Cell Suspensions using Nanoporous Membrane Electrodes.

A flow-through electroporation system, based on a novel nanoporous membrane/electrode design, for the delivery of cell wall-impermeant molecules into model leukocytes, HL-60 promyelocytes, was demonstrated. The ability to apply low voltages to cell populations, with nm-scale concentrated electric field in a periodic array, contributes to high cell viability. With applied biases of 1-4V, delivery of target molecules was achieved with 90% viability and up to 65% transfection efficiency. More importantly, the system allowed electrophoretic pumping of molecules from a microscale reservoir across the membrane/electrode system into a microfluidic flow channel for transfection of cells, a design that can reduce reagent amount by eightfold compared to current strategies. The flow-through system, which forces intimate membrane/electrode contact by using a 10μm channel height, can be easily scaled-up by adjusting the microfluidic channel geometry and/or the applied voltage pulse frequency to control cell residence times at the cell membrane/electrode interface. The demonstrated system shows promise in clinical applications where low-cost, high cell viability and high volume transfection methods are needed without the risk of viral vectors. In particular genetic modification of freely mobile white blood cells to either target disease cells or to express desired protein/enzyme biomolecules is an important target platform enabled by this device system.

603. Feldan Shuttle - Advanced Protein Delivery Technology for Cell Therapy

Methods for maximizing the therapeutic value of human cells often involve the transfer of genetic material and use of viruses, both raising important safety and economical concerns. One promising solution to improve cell therapy processes consists on the direct delivery of active proteins into human cells. Regenerative medicine studies established powerful proofs of concept using delivery of transcription factors for cell reprogramming and differentiation; however, the lack of efficiency of current protein delivery methods slowed down the transfer of these methods toward human clinical researches. In the last years, the CRISPR/Cas9 revolution brought forward the possibility of using diverse transfection approaches to deliver Cas9 protein complexed with guide RNA, a method yielding interesting results with cationic lipid agents and electroporation systems. In a human therapy context, lipidic agents are generally not suitable because of their high cellular toxicity. As for electroporation, few systems have been approved for cell therapy; in addition, this process represents a serious concern because of its high cost, inconsistency, associated cell mortality and the fact that some cells are simply refractory to electroporation. In all, these characteristics accentuate the need for a protein delivery process suitable for human cell therapy. In order to efficiently use transcription factors, Cas9 or any other intracellular proteins in cell therapy, Feldan Therapeutics is developing a protein delivery agent, the Feldan Shuttle, explicitly designed for cell therapy. Feldan Shuttle is entirely protein-based, thus allowing cells to naturally degrade it and the delivered protein after their active use. With this novel method, Feldan Therapeutics successfully delivered fluorescent proteins as well as functionally active transcription factors and Cas9/RNA complex with high efficiency and low toxicity into multiple cell lines and human primary cells. With this new technology, Feldan Therapeutics offers an innovative method designed to efficiently and safely deliver native proteins for cell therapy applications.

Preclinical evaluation of an endoscopic electroporation system.

Targeted delivery of specific chemotherapeutic drugs into tumors can be achieved by delivering electrical pulses directly to the tumor tissue. This causes a transient formation of pores in the cell membrane that enables passive diffusion of normally impermeant drugs. A novel device has been developed to enable the endoscopic delivery of this tumor permeabilizing treatment. The aim of the preclinical studies described here was to investigate the efficacy and safety of this nonthermal ablation system in the treatment of gastrointestinal cancer models. Murine, porcine, and canine gastrointestinal tumors and tissues were used to assess the efficacy and safety of electroporation delivered through the special device in combination with bleomycin. Tumor cell death, volume, and overall survival were recorded. Murine tumors treated with electrochemotherapy showed excellent responses, with cell death being induced rapidly, mainly via an apoptotic-type mechanism. Use of the system in canine gastrointestinal cancers demonstrated successful local endoluminal tumor resolution, with no safety or adverse effects noted. Electroporation via the new device in combination with bleomycin offers a nonthermal tumor ablative approach, and presents clinicians with a new option for the management of gastrointestinal cancers.

A novel electroporation system for efficient molecular delivery into Chlamydomonas reinhardtii with a 3-dimensional microelectrode.

Electroporation is one of the most widely used transfection methods because of its high efficiency and convenience among the various transfection methods. Previous micro-electroporation systems have some drawbacks such as limitations in height and design, time-consuming and an expensive fabrication process due to technical constraints. This study fabricates a three dimensional microelectrode using the 3D printing technique. The interdigitated microstructure consisting of poly lactic acid was injected by a 3D printer and coated with silver and aluminum with a series of dip-coatings. With the same strength of electric field (V cm−1), a higher efficiency for molecular delivery and a higher cellular viability are achieved with the microelectrode than with a standard cuvette. In addition, this study investigates chemicophysical changes such as Joule heating and dissolved metal during electroporation and showed the micro-electroporation system had less chemicophysical changes. It was concluded that the proposed micro-electroporation system will contribute to genetic engineering as a promising delivery tool, and this combination of 3D printing and electroporation has many potential applications for diverse designs or systems.

디지털 미세유체를 이용한 미세녹조류 형질전환에서의 세포벽의 영향 분석

Digital microfluidic electroporation system was used for the transformation of microalgae and we have obtained higher transformation efficiency and viability than that of conventional method. Key parameters of electroporation such as pulse voltage, number, and duration time were systematically investigated for two different microalgal strains with and without cell wall. We have found that cell wall does not always have negative effects on the gene transformation of microalgae. Parallel processing of proposed digital microfluidic electroporation was demonstrated together with on chip culture of microalgae.

Digital Microfluidic Approach for Efficient Electroporation with High Productivity: Transgene Expression of Microalgae without Cell Wall Removal.

A unique digital microfluidic electroporation (EP) system successfully demonstrates higher transgene expression than that of conventional techniques, in addition to reliable productivity and feasible integrated processes. By systematic investigations into the effects of the droplet EP conditions for a wild-type microalgae, 1 order of magnitude higher transgene expression is accomplished without cell wall removal over the conventional bulk EP system. In addition, the newly proposed droplet EP method by a droplet contact charging phenomena shows a great potential for the integration of EP processes and on-chip cell culture providing easy controllability of each process. Finally, the implications of the accomplishments and future directions for development of the proposed technology are discussed.

298. Transfection of Neutrophils from X-Linked Chronic Granulomatous Disease Patients with gp91phox mRNA Restores Oxidase Activity with High Efficiency and Viability

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to defective phagocyte NADPH oxidase required for production of antimicrobial oxidants. Fungal or bacterial infections in CGD may persist despite intensive and prolonged antibiotic therapy. In settings where conventional antibiotic therapy and/or surgical debridement do not control infection, use of allogeneic unmatched donor granulocyte transfusions has been therapeutic. However, CGD patients treated with granulocyte transfusions may develop high titers of anti-HLA antibodies, restricting use of allogeneic granulocyte transfusions and further complicating potentially curative bone marrow transplant. Using the CGD patient as an autologous donor for granulocyte transfusions would be a helpful solution.Here we demonstrate highly efficient functional correction of the functional defect in CGD by transfection of patient's own granulocytes with mRNA while retaining viability using a scalable, cGMP-compliant MaxCyte® GT™ electroporation system capable of handling clinically relevant numbers of cells (>1010cells).Freshly collected granulocytes from normal volunteers were electroporated using the MaxCyte electroporation system with eGFP-mRNA on the day of collection. Flow cytometry of demonstrated that >90% of neutrophils expressed eGFP with viability exceeding 90% at 24 hrs after electroporation. Phagocyte NADPH oxidase activity of the normal neutrophils was retained as well as bactericidal activity against Burkolderia cepacia, a CGD bacterial pathogen. Antibacterial activity of electroporated granulocytes was comparable to unmanipulated granulocytes confirming retention of cellular function following the electroporation. Finally, electroporated eGFP positive human neutrophils injected into CGD mice were shown to remain in circulation as well as migrate to the peritoneal cavity following thioglycollate IP injection and there were no adverse effects observed in the mice.Electroporation of gp91phox mRNA into human X-linked CGD (gp91phox-deficient) patient neutrophils resulted in expression of gp91phox protein in >70% of cells with >90% viability at 24 hours after treatment. Using the dihydrorhodamine assay of phagocyte NADPH oxidase activity, >70% of neutrophils remained functionally corrected 2 days after transfection. Other studies of cellular function in vitro and in vivo with electroporated corrected CGD neutrophils are in progress. In summary, we show the feasibility of restoring the function of CGD patient-autologous neutrophils via electroporation of mRNA encoding the defective gene product. This approach may have potential clinical utility in the management of severe chronic bacterial and fungal infections in CGD but also short-term correction of other hematopoietic gene diseases.

54. Genome Editing of Primary Human CD34+ Hematopoietic Stem Cells Enables a Safe Harbor Targeted Gene Addition Therapeutic Strategy for Chronic Granulomatous Disease

Many monogenic recessive diseases of blood can, in principle, be cured by transfer of functional therapeutic transgene to the genome of the hematopoietic stem cell (HSC) – a strategy proven successful for multiple rare diseases using current integrating vector gene therapy. With a focus on X-linked chronic granulomatous disease (X-CGD), we report a directed approach orthogonal to randomly integrating retrovector gene therapy: the highly specific targeted placement of the curative transgene into a validated safe harbor locus in human HSCs via human genome editing with zinc finger nucleases (ZFNs) and donor insert delivery using an AAV6 vector.We describe here an integrated targeted delivery platform customized for targeted addition to human HSCs using a cGMP-compliant electroporation system compatible with clinical scale production. Using next-generation, highly optimized ZFNs against the AAVS1/PPP1R12C gene locus, we optimized conditions for addition of the fluorescent Venus cDNA into human peripheral blood G-CSF mobilized CD34+ HSCs. Venus expression in manipulated human HSCs in vitro reached >50% efficiency, while earlier experiments demonstrated persistence of gene-modified cells in NSG mice with 12-15% Venus+ human CD45+ cells retrieved from transplanted mouse bone marrow and overall human HSC engraftment levels of >15%. Targeted integration (TI) rates achieved in human CD45+ cells from mouse bone marrow were 28-57%. Similar levels of Venus+ (~10%) are observed in spleen and peripheral blood CD45+ cells, indicating differentiation of gene-modified CD34 HSCs into circulating blood cells.X-CGD patients suffer from severe bacterial and fungal infections with excessive inflammation due to a defect in the gp91phox subunit of phagocyte oxidase. To extend the results above to CGD, we therefore used the same approach to target addition of the relevant curative transgene, gp91phox, into the AAVS1 safe harbor locus of HSCs from patients with X-linked CGD. In vitro levels of gp91phox expression in gene-modified patient CD34+ HSCs population achieve 12-16% gp91phox expression by flow cytometric analysis, with an NSG xenograft study demonstrating 3-5% of the engrafted human CD45+ cells expressing gp91phox. Of note, the MND-driven gp91phox expression from the safe harbor locus in human neutrophils differentiating from CD34+ cells transplanted into the NSG mouse model parallels wildtype gp91phox levels produced at the native locus.Our studies demonstrate the feasibility of targeted addition of different genes at the AAVS1 safe harbor site of the genome in human HSCs at an unprecedented efficiency and specificity; we demonstrate the efficient correction of the enzymatic defect in neutrophils arising from patient-derived HSCs in vivo. In sum with the advances in GMP-scale cell processing for genome editing, and the charted regulatory path for ZFNs to the clinic provided by ongoing trials in HIV, our studies represent the foundation for a rapid translation of ZFN-driven targeted addition as a clinical modality for X-linked CGD.