A Multi-Frequency 3D Printed Hand Phantom for Electromagnetic Measurements.

It has been shown that the presence of a hand holding a wireless handset (cell phone) can influence antenna efficiency and the measurement of specific absorption rate (SAR) and electromagnetic compatibility. Head phantoms, used in handset compliance testing to estimate SAR in the head, have achieved low cost and multi-frequency use. Head phantoms typically consist of a thin plastic shell, open on the top, holding a tissue simulating fluid. The specific simulant fluid used is determined by the radio frequency of the test. IEC 62209-1 has recipes, using safe nontoxic materials, for all the required frequency bands. Thus, head phantoms can be reused at different frequencies simply by changing the tissue simulating fluid. However, standards have not adopted the use of hand phantoms because SAR limits in limbs are less restrictive than the head, the tissue depth in a hand is insufficient to make accurate measurements with current electric field probes, and the cost of a solid hand phantom is limited to a single frequency band. Our goal was to determine whether 3D printing techniques would allow the construction of a hand phantom with the same utility as existing head phantoms. We developed this phantom based on computer simulations to determine how much human anatomy needed to be included in the phantom to obtain results consistent with actual use. Electric field scans of a handset alone, and held by the hand phantom, were performed. Comparison of handset scans using the phantom and human subjects was planned, but not performed due to Covid-19 restrictions and subsequent changes in priorities. We feel a fluid-filled 3D printed hand phantom is viable and practical. The 3D print files are available on GitHub.

Static magnetic field measurements of smart phones and watches and applicability to triggering magnet modes in implantable pacemakers and implantable cardioverter-defibrillators.

BACKGROUND: Implantable pacemakers and implantable cardioverter-defibrillators (ICDs) are designed to include a "magnet mode" feature that can be activated from magnets stronger than 10 G. This feature is designed to be used when a patient is undergoing a procedure where electromagnetic interference is possible, or anytime suspension of tachycardia detection and therapy is needed. A publication in Heart Rhythm demonstrates an iPhone 12 triggering the magnet mode of a Medtronic ICD. OBJECTIVE: The purpose of this study is to determine the separation distance between consumer electronic devices that may create magnetic interference, including cell phones and smart watches, and implantable pacemakers and ICDs where magnet mode can be triggered. METHODS: The static magnetic fields of the iPhone 12 models and Apple Watch were measured at several planes in 1 cm resolution using an FW Bell 5180 Gauss Meter with STD18-0404 Transverse probe (unidirectional probe). RESULTS: All iPhone 12 and Apple Watch 6 models tested have static magnetic fields significantly greater than 10 G in close proximity (1-11 mm), which attenuates to below 10 G between 11 and 20 mm. CONCLUSION: The findings of this study support the US Food and Drug Administration recommendation that patients keep any consumer electronic devices that may create magnetic interference, including cell phones and smart watches, at least 6 inches away from implanted medical devices, in particular pacemakers and cardiac defibrillators.

Preclinical studies of efficacy thresholds and tolerability of a clinically ready lentiviral vector for pyruvate kinase deficiency treatment.

Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene. PKD is characterized by non-spherocytic hemolytic anemia of variable severity and may be fatal in some cases during early childhood. Although not considered the standard of care, allogeneic stem cell transplantation has been shown as a potentially curative treatment, limited by donor availability, toxicity, and incomplete engraftment. Preclinical studies were conducted to define conditions to enable consistent therapeutic reversal, which were based on our previous data on lentiviral gene therapy for PKD. Improvement of erythroid parameters was identified by the presence of 20%-30% healthy donor cells. A minimum vector copy number (VCN) of 0.2-0.3 was required to correct PKD when corrected cells were transplanted in a mouse model for PKD. Biodistribution and pharmacokinetics studies, with the aim of conducting a global gene therapy clinical trial for PKD patients (RP-L301-0119), demonstrated that genetically corrected cells do not confer additional side effects. Moreover, a clinically compatible transduction protocol with mobilized peripheral blood CD34+ cells was optimized, thus facilitating the efficient transduction on human cells capable of repopulating the hematopoiesis of immunodeficient mice. We established conditions for a curative lentiviral vector gene therapy protocol for PKD.

Geochemical and Stable Fe Isotopic Analysis of Dissimilatory Microbial Iron Reduction in Chocolate Pots Hot Spring, Yellowstone National Park.

Chocolate Pots hot spring (CP) is an Fe-rich, circumneutral-pH geothermal spring in Yellowstone National Park. Relic hydrothermal systems have been identified on Mars, and modern hydrothermal environments such as CP are useful for gaining insight into potential pathways for generation of biosignatures of ancient microbial life on Earth and Mars. Fe isotope fractionation is recognized as a signature of dissimilatory microbial iron oxide reduction (DIR) in both the rock record and modern sedimentary environments. Previous studies in CP have demonstrated the presence of DIR in vent pool deposits and show aqueous-/solid-phase Fe isotope variations along the hot spring flow path that may be linked to this process. In this study, we examined the geochemistry and stable Fe isotopic composition of spring water and sediment core samples collected from the vent pool and along the flow path, with the goal of evaluating whether Fe isotopes can serve as a signature of past or present DIR activity. Bulk sediment Fe redox speciation confirmed that DIR is active within the hot spring vent pool sediments (but not in more distal deposits), and the observed Fe isotope fractionation between Fe(II) and Fe(III) is consistent with previous studies of DIR-driven Fe isotope fractionation. However, modeling of sediment Fe isotope distributions indicates that DIR does not produce a unique Fe isotopic signature of DIR in the vent pool environment. Because of rapid chemical and isotopic communication between the vent pool fluid and sediment, sorption of Fe(II) to Fe(III) oxides would produce an isotopic signature similar to DIR despite DIR-driven generation of large quantities of isotopically light solid-associated Fe(II). The possibility exists, however, for preservation of specific DIR-derived Fe(II) minerals such as siderite (which is present in the vent pool deposits), whose isotopic composition could serve as a long-term signature of DIR in relic hot spring environments.

Magnesium isotope analysis of olivine and pyroxene by SIMS: Evaluation of matrix effects.

The performance of multi-collector secondary ion mass spectrometry (MC-SIMS) for Mg isotope ratio analysis was evaluated using 17 olivine and 5 pyroxene reference materials (RMs). The Mg isotope composition of these RMs was accurately and precisely determined by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS), and these measured isotope ratios were used to evaluate SIMS instrumental mass bias as a function of the forsterite (Fo) content of olivine. The magnitude of the Mg isotope matrix effects were ~3‰ in δ25Mg, and are a complex function of olivine Fo content, that ranged from Fo59.3 to Fo100. In addition to these Mg isotope matrix effects, Si+ ion yields and Mg+/Si+ ion ratios varied as a complex function of the Fo content of the olivine RMs. For example, Si+ ion yields varied by ~33%. Based on the observations, we propose instrumental bias correction procedures for SIMS Mg isotope analysis of olivine using a combination of Mg+/Si+ ratios and Fo content of olivine. Using this correction method, the accuracy of δ25Mg analyses is 0.3‰, except for analysis of olivine with Fo86-88 where instrumental biases and Mg+/Si+ ratios change dramatically with Fo content, making it more difficult to assess the accuracy of Mg isotope ratio measurements by SIMS over this narrow range of Fo content. Five pyroxene RMs (3 orthopyroxenes and 2 clinopyroxenes) show smaller ranges of instrumental bias (~1.4‰ in δ25Mg) as compared to the olivine RMs. The instrumental bias for the 3 orthopyroxene RMs do not define a linear relationship with respect to enstatite (En) content, that ranged from En85.5 -96.3. The clinopyroxene RMs have similar En and wollastonite (Wo) contents but have δ25Mg values that differ by 0.5‰ relative to their δ25Mg values determined by MC-ICP-MS. These results indicate that additional factors (e.g., minor element abundances) likely contribute to SIMS instrumental mass fractionation. In order to better correct for these SIMS matrix effects, additional pyroxene RMs with various chemical compositions and known Mg isotope ratios are needed.

K isotopes as a tracer for continental weathering and geological K cycling.

The causal effects among uplift, climate, and continental weathering cannot be fully addressed using presently available geochemical proxies. However, stable potassium (K) isotopes can potentially overcome the limitations of existing isotopic proxies. Here we report on a systematic investigation of K isotopes in dissolved load and sediments from major rivers and their tributaries in China, which have drainage basins with varied climate, lithology, and topography. Our results show that during silicate weathering, heavy K isotopes are preferentially partitioned into aqueous solutions. Moreover, δ41K values of riverine dissolved load vary remarkably and correlate negatively with the chemical weathering intensity of the drainage basin. This correlation allows an estimate of the average K isotope composition of global riverine runoff (δ41K = -0.22‰), as well as modeling of the global K cycle based on mass balance calculations. Modeling incorporating K isotope mass balance better constrains estimated K fluxes for modern global K cycling, and the results show that the δ41K value of seawater is sensitive to continental weathering intensity changes. Thus, it is possible to use the δ41K record of paleo-seawater to infer continental weathering intensity through Earth's history.

Minimal conditioning in Fanconi anemia promotes multi-lineage marrow engraftment at 10-fold lower cell doses.

BACKGROUND: Gene therapy approaches for the treatment of Fanconi anemia (FA) hold promise for patients without a suitably matched donor for an allogeneic bone marrow transplant. However, significant limitations include the collection of sufficient stem cell numbers from patients, the fragility of these cells during ex vivo manipulation, and clinically meaningful engraftment following transplantation. With these challenges in mind, we were interested in determining (i) whether gene-corrected cells at progressively lower numbers can successfully engraft in FA; (ii) whether low-dose conditioning facilitates this engraftment; and (iii) whether these cells can be selected for post-transplant. METHODS: Utilizing a well characterized mouse model of FA, we infused donor bone marrow from healthy heterozygote littermates that are unaffected carriers of the FANCA mutation to mimic a gene-corrected product, after administering low-dose conditioning. Once baseline engraftment was observed, we administered a second, very-low selective dose to determine whether gene-corrected cells could be selected for in vivo. RESULTS: We demonstrate that upfront low-dose conditioning greatly increases successful engraftment of hematopoietic corrected cells in a pre-clinical animal model of FA. Additionally, without conditioning, cells can still engraft and demonstrate a selective advantage in vivo over time following transplantation, and these corrected cells can be directly selected for in vivo after engraftment. CONCLUSIONS: Minimal conditioning prior to bone marrow transplant in Fanconi anemia promotes the multi-lineage engraftment of 10-fold fewer cells compared to nonconditioned controls. These data provide important insights into the potential of minimally toxic conditioning protocols for FA gene therapy applications.

Two billion years of magmatism recorded from a single Mars meteorite ejection site.

The timing and nature of igneous activity recorded at a single Mars ejection site can be determined from the isotope analyses of Martian meteorites. Northwest Africa (NWA) 7635 has an Sm-Nd crystallization age of 2.403 ± 0.140 billion years, and isotope data indicate that it is derived from an incompatible trace element-depleted mantle source similar to that which produced a geochemically distinct group of 327- to 574-million-year-old "depleted" shergottites. Cosmogenic nuclide data demonstrate that NWA 7635 was ejected from Mars 1.1 million years ago (Ma), as were at least 10 other depleted shergottites. The shared ejection age is consistent with a common ejection site for these meteorites. The spatial association of 327- to 2403-Ma depleted shergottites indicates >2 billion years of magmatism from a long-lived and geochemically distinct volcanic center near the ejection site.

Iron Isotope Fractionations Reveal a Finite Bioavailable Fe Pool for Structural Fe(III) Reduction in Nontronite.

We report on stable Fe isotope fractionation during microbial and chemical reduction of structural Fe(III) in nontronite NAu-1. (56)Fe/(54)Fe fractionation factors between aqueous Fe(II) and structural Fe(III) ranged from -1.2 to +0.8‰. Microbial (Shewanella oneidensis and Geobacter sulfurreducens) and chemical (dithionite) reduction experiments revealed a two-stage process. Stage 1 was characterized by rapid reduction of a finite Fe(III) pool along the edges of the clay particles, accompanied by a limited release to solution of Fe(II), which partially adsorbed onto basal planes. Stable Fe isotope compositions revealed that electron transfer and atom exchange (ETAE) occurred between edge-bound Fe(II) and octahedral (structural) Fe(III) within the clay lattice, as well as between aqueous Fe(II) and structural Fe(III) via a transient sorbed phase. The isotopic fractionation factors decreased with increasing extent of reduction as a result of the depletion of the finite bioavailable Fe(III) pool. During stage 2, microbial reduction was inhibited while chemical reduction continued. However, further ETAE between aqueous Fe(II) and structural Fe(III) was not observed. Our results imply that the pool of bioavailable Fe(III) is restricted to structural Fe sites located near the edges of the clay particles. Blockage of ETAE distinguishes Fe(III) reduction of layered clay minerals from that of Fe oxyhydroxides, where accumulation of structural Fe(II) is much more limited.

Creation of zebularine-resistant human cytidine deaminase mutants to enhance the chemoprotection of hematopoietic stem cells.

Human cytidine deaminase (hCDA) is a biomedically important enzyme able to inactivate cytidine nucleoside analogs such as the antileukemic agent cytosine arabinoside (AraC) and thereby limit antineoplastic efficacy. Potent inhibitors of hCDA have been developed, e.g. zebularine, that when administered in combination with AraC enhance antineoplastic activity. Tandem hematopoietic stem cell (HSC) transplantation and combination chemotherapy (zebularine and AraC) could exhibit robust antineoplastic potency, but AraC-based chemotherapy regimens lead to pronounced myelosuppression due to relatively low hCDA activity in HSCs, and this approach could exacerbate this effect. To circumvent the pronounced myelosuppression of zebularine and AraC combination therapy while maintaining antineoplastic potency, zebularine-resistant hCDA variants could be used to gene-modify HSCs prior to transplantation. To achieve this, our approach was to isolate hCDA variants through random mutagenesis in conjunction with selection for hCDA activity and resistance to zebularine in an Escherichia coli genetic complementation system. Here, we report the identification of nine novel variants from a pool of 1.6 × 106 transformants that conferred significant zebularine resistance relative to wild-type hCDA2. Several variants revealed significantly higher Ki values toward zebularine when compared with wild-type hCDA values and, as such, are candidates for further exploration for gene-modified HSC transplantation approaches.

VISA--Vector Integration Site Analysis server: a web-based server to rapidly identify retroviral integration sites from next-generation sequencing.

BACKGROUND: Analyzing the integration profile of retroviral vectors is a vital step in determining their potential genotoxic effects and developing safer vectors for therapeutic use. Identifying retroviral vector integration sites is also important for retroviral mutagenesis screens. RESULTS: We developed VISA, a vector integration site analysis server, to analyze next-generation sequencing data for retroviral vector integration sites. Sequence reads that contain a provirus are mapped to the human genome, sequence reads that cannot be localized to a unique location in the genome are filtered out, and then unique retroviral vector integration sites are determined based on the alignment scores of the remaining sequence reads. CONCLUSIONS: VISA offers a simple web interface to upload sequence files and results are returned in a concise tabular format to allow rapid analysis of retroviral vector integration sites.

Biologically recycled continental iron is a major component in banded iron formations.

Banded iron formations (BIFs) record a time of extensive Fe deposition in the Precambrian oceans, but the sources and pathways for metals in BIFs remain controversial. Here, we present Fe- and Nd-isotope data that indicate two sources of Fe for the large BIF units deposited 2.5 billion y ago. High-εNd and -δ(56)Fe signatures in some BIF samples record a hydrothermal component, but correlated decreases in εNd- and δ(56)Fe values reflect contributions from a continental component. The continental Fe source is best explained by Fe mobilization on the continental margin by microbial dissimilatory iron reduction (DIR) and confirms for the first time, to our knowledge, a microbially driven Fe shuttle for the largest BIFs on Earth. Detailed sampling at various scales shows that the proportions of hydrothermal and continental Fe sources were invariant over periods of 10(0)-10(3) y, indicating that there was no seasonal control, although Fe sources varied on longer timescales of 10(5)-10(6) y, suggesting a control by marine basin circulation. These results show that Fe sources and pathways for BIFs reflect the interplay between abiologic (hydrothermal) and biologic processes, where the latter reflects DIR that operated on a basin-wide scale in the Archean.

(211)Astatine-Conjugated Monoclonal CD45 Antibody-Based Nonmyeloablative Conditioning for Stem Cell Gene Therapy.

Most hematopoietic stem cell gene therapy studies require host conditioning to allow for efficient engraftment of gene-modified cells. Conditioning regimens with lower treatment-related toxicities are especially relevant for the treatment of nonmalignant blood disorders, such as hemoglobinopathies and immunodeficiencies, and for patients who are otherwise ineligible for conventional high-dose conditioning. Radioimmunotherapy, which employs an α- or a ß-emitting radionuclide conjugated to a targeting antibody, is effective for delivering cytotoxic doses of radiation to a cell type of interest while minimizing off-target toxicity. Here, we demonstrate the feasibility of using a nonmyeloablative dose of a monoclonal anti-CD45 antibody conjugated to the α-emitter Astatine-211 ((211)At) to promote engraftment of an autologous gene-modified stem cell graft in the canine model. The doses used provided myelosuppression with rapid autologous recovery and minimal off-target toxicity. Engraftment levels were low in all dogs and reflected the low numbers of gene-modified cells infused. Our data suggest that a cell dose exceeding 1×10(6) cells/kg be used with nonmyeloablative doses of (211)At-anti-CD45 monoclonal antibodies for sustained engraftment in the dog model.

Atom exchange between aqueous Fe(II) and structural Fe in clay minerals.

Due to their stability toward reductive dissolution, Fe-bearing clay minerals are viewed as a renewable source of Fe redox activity in diverse environments. Recent findings of interfacial electron transfer between aqueous Fe(II) and structural Fe in clay minerals and electron conduction in octahedral sheets of nontronite, however, raise the question whether Fe interaction with clay minerals is more dynamic than previously thought. Here, we use an enriched isotope tracer approach to simultaneously trace Fe atom movement from the aqueous phase to the solid ((57)Fe) and from the solid into the aqueous phase ((56)Fe). Over 6 months, we observed a significant decrease in aqueous (57)Fe isotope fraction, with a fast initial decrease which slowed after 3 days and stabilized after about 50 days. For the aqueous (56)Fe isotope fraction, we observed a similar but opposite trend, indicating that Fe atom movement had occurred in both directions: from the aqueous phase into the solid and from the solid into aqueous phase. We calculated that 5-20% of structural Fe in clay minerals NAu-1, NAu-2, and SWa-1 exchanged with aqueous Fe(II), which significantly exceeds the Fe atom layer exposed directly to solution. Calculations based on electron-hopping rates in nontronite suggest that the bulk conduction mechanism previously demonstrated for hematite1 and suggested as an explanation for the significant Fe atom exchange observed in goethite2 may be a plausible mechanism for Fe atom exchange in Fe-bearing clay minerals. Our finding of 5-20% Fe atom exchange in clay minerals indicates that we need to rethink how Fe mobility affects the macroscopic properties of Fe-bearing phyllosilicates and its role in Fe biogeochemical cycling, as well as its use in a variety of engineered applications, such as landfill liners and nuclear repositories.

RF Interference in Hearing Aids from Cellphones Part 1: Near-field cellphone emissions measurements and the effects of hands.

Cellular telephones (cellphones) are currently categorized for hearing aid compatibility based on a calculated value (metric) obtained from the measurement of near-field, radio-frequency emissions according to a procedure described in ANSI Standard C63.19 "Measurement of Compatibility between Wireless Communications Devices and Hearing Aids". There has been a lack of documentation, however, that relates this metric to a cellphone's potential for interference in actual use, that is, when it is held at the ear in a normal-use position by a hearing aid wearer. In Part 1 of this two-part series, we compare the ANSI C63.19 metric to simpler metrics, still based on the near-field test procedure of the standard, and to near-field measurements made when the cellphones are hand-held. The results justify employing a simpler no-hand metric than the exclusion area procedure presently specified by the standard, but not the addition of a test hand to the procedure. The further effect of the head and interaction with the hearing aid is examined in Part 2 of the series.

RF Interference in Hearing Aids from Cellphones Part 2: Comparing in-use RF coupling to predictions.

Cellphones and hearing aids are presently tested for their near-field RF emissions and RF immunity, respectively, to predict their mutual compatibility when used together. In the concluding part of this two-part series, we examine the relationship between these independent device measurements and the resultant in-use coupled RF interference, which may be heard as audio frequency noises by the hearing aid wearer. The established standards are seen to be generally reasonable in meeting the compatibility goals (i.e., ensuring a low level of perceived audio interference), but the combined effects of the relative device positioning, the hand, and especially the head add a high degree of uncertainty to the relationship between the actual in-use RF interference coupling and predictions based on individual emissions and immunity measurements.

Fe(II)-catalyzed recrystallization of goethite revisited.

Results from enriched (57)Fe isotope tracer experiments have shown that atom exchange can occur between structural Fe in Fe(III) oxides and aqueous Fe(II) with no formation of secondary minerals or change in particle size or shape. Here we derive a mass balance model to quantify the extent of Fe atom exchange between goethite and aqueous Fe(II) that accounts for different Fe pool sizes. We use this model to reinterpret our previous work and to quantify the influence of particle size and pH on extent of goethite exchange with aqueous Fe(II). Consistent with our previous interpretation, substantial exchange of goethite occurred at pH 7.5 (≈ 90%) and we observed little effect of particle size between nanogoethite (average size of 81 × 11 nm; ≈ 110 m(2)/g) and microgoethite (average size of 590 × 42 nm; ≈ 40 m(2)/g). Despite ≈ 90% of the bulk goethite exchanging at pH 7.5, we found no change in mineral phase, average particle size, crystallinity, or reactivity after reaction with aqueous Fe(II). At a lower pH of 5.0, no net sorption of Fe(II) was observed and significantly less exchange occurred accounting for less than the estimated proportion of surface Fe atoms in the particles. Particle size appears to influence the amount of exchange at pH 5.0 and we suggest that aggregation and surface area may play a role. Results from sequential chemical extractions indicate that (57)Fe accumulates in extracted Fe(III) goethite components. Isotopic compositions of the extracts indicate that a gradient of (57)Fe develops within the goethite with more accumulation of (57)Fe occurring in the more easily extracted Fe(III) that may be nearer to the surface.

Gene therapy enhances chemotherapy tolerance and efficacy in glioblastoma patients.

BACKGROUND: Temozolomide (TMZ) is one of the most potent chemotherapy agents for the treatment of glioblastoma. Unfortunately, almost half of glioblastoma tumors are TMZ resistant due to overexpression of methylguanine methyltransferase (MGMT(hi)). Coadministration of O6-benzylguanine (O6BG) can restore TMZ sensitivity, but causes off-target myelosuppression. Here, we conducted a prospective clinical trial to test whether gene therapy to confer O6BG resistance in hematopoietic stem cells (HSCs) improves chemotherapy tolerance and outcome. METHODS: We enrolled 7 newly diagnosed glioblastoma patients with MGMT(hi) tumors. Patients received autologous gene-modified HSCs following single-agent carmustine administration. After hematopoietic recovery, patients underwent O6BG/TMZ chemotherapy in 28-day cycles. Serial blood samples and tumor images were collected throughout the study. Chemotherapy tolerance was determined by the observed myelosuppression and recovery following each cycle. Patient-specific biomathematical modeling of tumor growth was performed. Progression-free survival (PFS) and overall survival (OS) were also evaluated. RESULTS: Gene therapy permitted a significant increase in the mean number of tolerated O6BG/TMZ cycles (4.4 cycles per patient, P < 0.05) compared with historical controls without gene therapy (n = 7 patients, 1.7 cycles per patient). One patient tolerated an unprecedented 9 cycles and demonstrated long-term PFS without additional therapy. Overall, we observed a median PFS of 9 (range 3.5-57+) months and OS of 20 (range 13-57+) months. Furthermore, biomathematical modeling revealed markedly delayed tumor growth at lower cumulative TMZ doses in study patients compared with patients that received standard TMZ regimens without O6BG. CONCLUSION: These data support further development of chemoprotective gene therapy in combination with O6BG and TMZ for the treatment of glioblastoma and potentially other tumors with overexpression of MGMT. TRIAL REGISTRATION: Clinicaltrials.gov NCT00669669. FUNDING: R01CA114218, R01AI080326, R01HL098489, P30DK056465, K01DK076973, R01HL074162, R01CA164371, R01NS060752, U54CA143970.

High-throughput genomic mapping of vector integration sites in gene therapy studies.

Gene therapy has enormous potential to treat a variety of infectious and genetic diseases. To date hundreds of patients worldwide have received hematopoietic cell products that have been gene-modified with retrovirus vectors carrying therapeutic transgenes, and many patients have been cured or demonstrated disease stabilization as a result (Adair et al., Sci Transl Med 4:133ra57, 2012; Biffi et al., Science 341:1233158, 2013; Aiuti et al., Science 341:1233151, 2013; Fischer et al., Gene 525:170-173, 2013). Unfortunately, for some patients the provirus integration dysregulated the expression of nearby genes leading to clonal outgrowth and, in some cases, cancer. Thus, the unwanted side effect of insertional mutagenesis has become a major concern for retrovirus gene therapy. The careful study of retrovirus integration sites (RIS) and the contribution of individual gene-modified clones to hematopoietic repopulating cells is of crucial importance for all gene therapy studies. Supporting this, the US Food and Drug Administration (FDA) has mandated the careful monitoring of RIS in all clinical trials of gene therapy. An invaluable method was developed: linear amplification mediated-polymerase chain reaction (LAM-PCR) capable of analyzing in vitro and complex in vivo samples, capturing valuable genomic information directly flanking the site of provirus integration. Linking this method and similar methods to high-throughput sequencing has now made possible an unprecedented understanding of the integration profile of various retrovirus vectors, and allows for sensitive monitoring of their safety. It also allows for a detailed comparison of improved safety-enhanced gene therapy vectors. An important readout of safety is the relative contribution of individual gene-modified repopulating clones. One limitation of LAM-PCR is that the ability to capture the relative contribution of individual clones is compromised because of the initial linear PCR common to all current methods. Here, we describe an improved protocol developed for efficient capture, sequencing, and analysis of RIS that preserves gene-modified clonal contribution information. We also discuss methods to assess dominant/overrepresented gene-modified clones in preclinical and clinical models.