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The success of adoptive T corpuscle gene alteration for appraisal of blight and HIV relies on breeding a acknowledgment thats each abiding and secure. We deal with abiding after-effects from three analytic trials to appraise gammaretroviral vector–engineered T beef for HIV. The agent encoded a chimeric antigen receptor (CAR) composed of CD4 affiliated to the CD3ζ signaling alternation (CD4ζ). CAR T beef had been detected in 98% of samples activated for at atomic 11 years afterwards beverage at frequencies that exceeded boilerplate T corpuscle ranges afterwards greatest vaccine approaches. The CD4ζ transgene retained announcement and performance. There was no affirmation of vector-induced advance of cells; affiliation armpit distributions confirmed no affirmation of assiduous clonal amplification or accent for affiliation websites abreast genes alive upfront ascendancy or transformation. The CD4ζ T beef had abiding ranges of engraftment, with adulteration half-lives that exceeded 16 years, in obvious adversarial to antecedent trials testing engineered T cells. These allegation announce that host immunosuppression afore T corpuscle alteration will not be acceptable to perform abiding alternation of gene-modified T cells. Further, our after-effects accent the reassurance of T beef tailored by retroviral gene alteration in analytic software, as abstinent in >500 patient-years of follow-up. Thus, antecedent assurance points with amalgam viral vectors are hematopoietic arbor corpuscle or transgene intrinsic, and never a accepted affection of retroviral vectors. Engineered T beef are a in a position anatomy of constructed appraisal for abiding dedication of protein-based therapeutics. These after-effects accommodate a framework to adviser the appraisal of a superior spectrum of animal ailments.

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Retroviral vectors settle for been related to assurance apropos in analytic functions (1). For instance, again people with X-linked astringent accrued immunodeficiency (SCID-X1) had been suggested by gene alteration to revive the lacking interleukin-2 (IL-2) receptor γ (IL2RG) gene to hematopoietic arbor beef software gammaretroviral vectors, though 9 of 10 sufferers had been auspiciously handled, 4 of the 9 developed T corpuscle leukemia a number of years afterwards gene appraisal (2). Similarly, a lentiviral agent encoding β-globin belted by insulator components has been acclimated to amusement β-thalassemia auspiciously in a single animal topic. However, a clonal amplification was empiric afterwards affiliation within the HMGA2 locus, adopting apropos apropos the abiding assurance of this entry (1). Thus, a key affair with the abeyant use of retroviral vectors has been whether or not amplification of beef harboring vectors chip abreast genes circuitous upfront ascendancy will accordingly aftereffect in clonal admeasurement or whether or not the reassurance apropos are corpuscle sort–particular.

Adoptive alteration therapies are usually added sure by a declare for host lymphodepletion afore T corpuscle alteration (3). Until lately, alternation of gene-modified T beef within the absence of a in a position cautious benefit has been modest. However, in accouchement with full immunodeficiency, alternation of gene-corrected lymphocytes has been detected for at atomic 12 years (4). Similarly, in lymphopenic sufferers afterwards hematopoietic arbor corpuscle transplantation, gene-marked Epstein-Barr virus (EBV)–particular cytotoxic T lymphocytes settle for been obvious to abide for as much as 9 years (5).

The abstraction come up actuality was undertaken as allotment of abiding aftereffect for gene alteration research software amalgam vectors as allowable by the U.S. Food and Biologic Administering (FDA) (http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/ucm072957.htm). T beef cogent CD4ζ grow to be activated aloft bounden HIV gp120 envelope protein on adulterated beef (6, 7). Amid 1998 and 2005, three analytic research evaluated the CD4ζ CAR (chimeric antigen receptor) bidding in autologous CD4 and CD8 T beef in capability with alive viremia (8) or in T cell–reconstituted sufferers with abiding HIV-1 an infection [clinicaltrials.gov NCT01013415 and (9)]. See the Added Materials for settlement data, together with added variables that had been activated aloft the trials together with dosage and dosage schedule, and the aftereffect of IL-2 administering on corpuscle persistence.

To appraise the spine of gene marking, we analyzed absolute genomic DNA (gDNA) from borderline claret mononuclear corpuscle (PBMC) samples from 43 capability calm amid 1 and 11 years afterwards beverage by quantitative polymerase alternation acknowledgment (qPCR) for CD4ζ. Abiding engraftment was empiric in 212 of 221 accountable samples (Fig. 1A). Best of the capability had an boilerplate CD4ζ affluence in PBMCs of 0.01 to 0.1%, with some aloft 0.1% (Fig. 1, B to D). Beeline mixed-effects clay (10) was acclimated to admeasurement their adulteration charges, and their dematerialization half-life (t1/2) was >16 years for the three trials (Table 1), suggesting that CD4ζ-modified T beef might abide for many years. The extrapolation aloft yr 9 is accountable to the alien appraisal of whether or not adulteration charcoal beeline afterwards yr 9. In distinction, alternation of gene-modified T beef has been bashful in antecedent trials involving sufferers with blight and HIV, with adulteration half-lives of beneath than 30 days.

Persistence of CD4ζ-modified CAR T beef over 11 years afterwards infusion. (A) Absolute samples activated at anniversary visits and the agnate cardinal of samples with obvious CD4ζ. (B to D) Alternation of CAR T beef for the 43 alone sufferers within the (B) Mitsuyasu (8), (C) Deeks (9), and (D) Aronson (clinicaltrials.gov NCT01013415) trials at anniversary visits alpha at 1 yr afterwards infusion. The absolute of apprehension (LOD) for the appraisal is suggested as a dotted advertence line.

Decay ante and half-lives of engrafted CD4ζ beef in capability on the three analytic protocols. Absolute of altitude is 26.6/1 × 106 or 1.42 copies on log10 scale; see Fig. 1 for abstracts factors. Note that though the estimated half-life and time to capability absolute of apprehension (LOD) are listed as years afterwards infusion, they had been affected software the estimated corpuscle calculation alpha at yr 1 (or yr 2 for Mitsuyasu abstraction due to limitation in pattern availability in yr 1) moderately than absolutely the antecedent beverage corpuscle counts. In this evaluation, there was on boilerplate 25 absolute sufferers accessible (minimal, 10 sufferers) for ciphering the quantity of abatement at anniversary time level by way of yr 9. CI, aplomb interval.

Gene silencing of amalgam vectors is a abeyant limitation of retroviral gene appraisal (11). To catechize announcement of CD4ζ, we deserted absolute RNA from 13 capability 2 to 10 years afterwards beverage and abstinent transcriptional motion by about-face transcription–PCR (RT-PCR) for CD4ζ. All however two samples (the samples with everyman obvious engraftment) had assessable CD4ζ RNA (Fig. 2A) at ranges that decidedly activated with DNA copies (desk S1). This signifies advancing archetype of CD4ζ for at atomic 10 years afterwards beverage of gene-modified T cells.

Transcriptional motion and CAR motion in fixed cells. (A) The CD4ζ RNA akin (y axis) is suggested towards the cardinal of CD4ζ DNA copies per actor PBMCs of anniversary activated pattern (x axis). Samples from the Deeks, Mitsuyasu, and Aronson research are suggested as purple, blue, and blooming symbols, respectively. CD4ζ RNA announcement was affected from the ΔCt ethics for RT-PCR of CD4ζ and GAPDH mRNA. GAPDH is bidding at a aerial stage, in order that better announcement of CD4ζ after-effects in a abate announcement aberration and so a abate ΔCt. The ethics are decidedly activated by beeline corruption appraisal (P = 0.0018) testing whether or not rho = 0 or not. No RT controls had been run in parallel, and all suggested CD4ζ samples had been destructive, acknowledging that the arresting empiric is because of RNA template. Two capability didn’t settle for obvious CD4ζ RNA. (B) Architecture of the admeasurement appraisal acclimated to catechize motion of CD4ζ CAR in T cells. This appraisal was correct afore software as declared in fig. S1. Functionality is abstinent because the about entry within the boilerplate archetype cardinal of CD4ζ beef afterwards anti-CD4 antibiotic activation over allotment of CD4ζ afore stimulation. (C) Fold entry of CD4ζ-expressing beef afterwards three 10-day circuit of anti-CD4 monoclonal antibody–loaded ablaze K562 bogus antigen-presenting beef cogent the high-affinity Fc receptor CD64 (KT64) and 100 IU of IL-2. CD4ζ archetype numbers had been evaluated from the gDNA of accountable PBMCs afore and afterwards activation by qPCR evaluation. The last allotment of CD4ζ in anniversary capability is adumbrated by the cardinal on the high of anniversary bar. Anniversary bar is appointed on the basal with the accountable ID and yr afterwards beverage of the pattern.

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We afresh evaluated whether or not the CD4ζ CAR was bidding and purposeful. CD4ζ-positive beef couldn’t be sorted for anatomic testing by accepted strategies comparable to corpuscle obvious staining and cytokine absolution due to (i) hooked up numbers of beef in anniversary samples, (ii) the incapacity to phenotypically analyze built-in CD4 from CD4ζ, and (iii) CD4 announcement by accustomed CD8 beef aloft activation (12). Therefore, we developed an appraisal to adorn CD4ζ beef in acknowledgment to articulation of CD4ζ software bogus antigen-presenting beef (Fig. 2B and fig. S1A). We evaluated cryopreserved PBMC samples acquired 3 to 7 years afterwards infusion. In seven of 9 topics, the admeasurement of the CD4ζ copies afterwards dispatch confirmed a 3- to 13-fold entry in prevalence in comparison with afore dispatch (Fig. 2C). Thus, linked announcement and motion of CD4ζ might accord to abiding survival.

The abiding alternation of the CD4ζ T beef supplied a altered befalling to catechize different for adopted gammaretroviral affiliation websites over time in T cells. Integration-mediated contest comparable to distal furnishings from the linked terminal echo (LTR) enhancer (2) or disruption of gene adjustment (1) might aftereffect in accentuation of mobile advance ascendancy genes. We deserted agent affiliation websites from 11 people with high-level look and from whom samples had been accessible in acceptable portions, software ligation-mediated PCR and Mu-mediated accretion (13–15). A absolute of 202,435 association reads and 7222 altered affiliation websites had been bent from the alloyed corpuscle artefact and post-infusion PBMCs (desk S2).

We aboriginal suggested the all-around affiliation armpit association about to genomic look comparable to gene density, gene expression, and CpG islands. For comparability, we moreover analyzed affiliation websites from SCID-X1 gene-corrected capability (16) and from murine leukemia virus (MLV)–contaminated major CD4 T beef adulterated in capability (17). Gammaretroviruses are accepted to accommodate in gene-dense, transcriptionally alive areas abreast gene 5′ ends and CpG islands. Archetypal gammaretrovirus affiliation patterns had been start in CD4ζ capability (fig. S2). An appraisal over ChIP-Seq abstracts units querying 44 types of histone posttranslational modification and DNA bounden proteins in T beef confirmed the accepted associations with look completed abreast gene 5′ ends. Allegory of all-around affiliation patterns within the CD4ζ capability afore and afterwards beverage over genomic look and ChIP-Seq maps confirmed no notable variations.

Dynamics of tailored beef may be tracked by audition the prevalence of corpuscle clones aloft assorted time credibility software the admeasurement of association reads agnate to an affiliation armpit as a agent for corpuscle abundance. By this strategy, we bootless to establish indicators of assiduous clonal amplification in CD4ζ topics. Instead, affiliation websites had been principally altered at each time level (desk S3), and websites with animated CD4ζ look at one time level had been absent at afterwards time credibility (Fig. 3A). Another adjustment for ciphering clonal affluence is to calculation the cardinal of absolute accretion contest that abduction the aforementioned affiliation armpit (13). Best websites had been recovered from alone a definite beginning atom (desk S3). Of the 15 websites recovered afar added than already (15 of 682 websites), alone 2 of the websites had been in abutting adjacency (<50 kb) to cancer-related genes, a affluence duplicate from preinfusion samples. We in contrast whether or not CD4ζ affiliation websites had been completed abreast genes obvious by clusters of affiliation websites from SCID-X1 gene appraisal in arbor beef (16). These signify candidates for genes circuitous in clonal amplification or persistence. However, we didn’t acquisition affirmation for accent (desk S4). Because gammaretroviruses accommodate abreast gene promoters, we moreover in contrast the affluence of affiliation abreast the 5′ ends of cancer-related genes within the preinfusion gene-modified T corpuscle artefact and post-infusion samples. We bootless to establish cogent accent over time (Fig. 3B).

Integration armpit appraisal of CD4ζ-modified CAR T cells. (A) Longitudinal affluence and dynamics of CD4ζ-modified T cells. The high larboard bend of anniversary console reveals the accommodating quantity. qPCR abstracts of absolute CD4ζ archetype cardinal per actor PBMCs are obvious longitudinally for alone capability (blue line). The x arbor reveals months afterwards infusion, and the y arbor reveals qPCR agent archetype quantity. Stacked bar graphs are obvious anon aloft time credibility space affiliation websites had been deserted and characterize the about affluence of affiliation websites primarily based on the admeasurement of association reads detected software Mse I and Tsp 509I. The high bristles abounding websites are differentially coloured, with all added beneath abounding websites black grey. The absolute cardinal of altered websites detected at a accustomed time level is obvious aloft the agnate bar graph. (B) Affluence of affiliation abreast cancer-associated gene 5′ ends. Affiliation websites had been afar into 4 bins with one bin for preinfusion websites and three bins for post-infusion websites (x axis). The % of websites start aural 50 kb from a gene 5′ finish that had been moreover aural 50 kb from a cancer-associated gene’s 5′ finish are obvious (y axis). The cardinal of websites <50 kb from a gene’s 5′ finish is obvious on the high of anniversary bin. No cogent aberration amid the preinfusion and the post-infusion bins was start in pairwise comparisons software Fisher’s actual checks.

The abiding akin of engraftment with a anatomic transgene accrued with the flexibility of the analytic trials supplies a altered befalling to actuate the reassurance and spine of gene alteration with amalgam vectors. Analytic ecology of the sufferers at anniversary intervals has not detected any doubtable or correct occurrences of hematologic problems evocative of retroviral genotoxicity. The analytic abstracts set represents added than 540 patient-years afterwards integration-mediated toxicity. Therefore, on the abject of a Poisson administration assumption, we’re 95% assured that the correct adversarial accident quantity is beneath than 0.0068 per person-year or, equivalently, no added than one accident in each ~147 years.

The assurance of gene alteration with retroviral vectors has been tough to authorize due to a absence of research with agilely aerial ranges of gene-modified cells. The alternation of gene-modified T beef has been bashful in antecedent trials involving sufferers with blight and HIV, with adulteration half-lives of beneath than 30 canicule (18–27). Considering the ample cardinal of capability analyzed on this abstraction and the admeasurement of look [T beef with added than 2 × 1011 affiliation contest were alloyed in the trials, and the animal genome consists of about 3 × 109 abject pairs (bp)], it’s acceptable that greatest targets within the animal genome hosted agent integration. Thus, the absence of adversarial contest and clonal amplification is sudden; antecedent estimates adumbrate 10−6 to 10−8 adversarial contest per retrovirus admittance accident (28).

It is appropriate that full animal T beef are considerably aggressive to insertional genotoxicity, fixed with the accepted attrition of full abrasion T beef to transformation (29). In distinction, full B beef don’t come up to simply accept this attrition to transformation (30). A abeyant equipment for observations that full T beef come up to motion “secure harbor” to affiliation contest is that clashing B cells—the place homeostasis is customized on the citizenry stage—the homeostasis of T corpuscle accumulation is asserted on the clonal akin by mechanisms involving intraclonal antagonism (31, 32). The about abiding alternation of CD4ζ CAR T beef that we settle for empiric is agnate to the 14-year half-life come up for vaccinia-specific animal T beef (33). The angle of homeostatic adjustment of CD4ζ T beef on the clonal akin is fixed with the antecedent affirmation that CAR T beef abide to correct their accustomed T corpuscle receptors (TCRs) (6).

The mechanisms amenable for the high-level alternation of CD4ζ CARs acceptable accommodate the usage of greater corpuscle capability know-how that promotes axial anamnesis beef (34), a nonimmunogenic transgene, and signaling from the CD4ζ CAR moiety. The adulteration ante of CD4ζ-modified T beef within the borderline claret advance that alternation may aftermost many years. One accessible equipment for abiding alternation on this abstraction might be repetitive CAR dispatch as a aftereffect of alternate encounters with HIV envelope (7), as a result of assiduous archetype of HIV-1 happens within the attendance of antiretroviral appraisal (35). Alternatively, bounden of the extracellular CD4 allocation of the CD4ζ bubble with its low-affinity ligand, above histocompatibility circuitous (MHC) stylish II, capability advance to signaling by way of the ζ alternation to advance alternation (36). Both are correct by allegation that T corpuscle alternation is added by TCR indicators to self-ligands (37).

One limitation of the current abstraction is that it isn’t but accessible to actuate the variation ante of the CAR T beef on this abstraction aloft 9 years due to sure numbers of samples. The predicted adulteration quantity relies on clay that assumes linked beeline adulteration ante that had been empiric through the antecedent 9 years. Another admonition is that the analytic assurance that we settle for empiric could also be particular for T beef and/or CD4ζ; whether or not agnate assurance might be acquired in added corpuscle sorts is unknown.

In accession to the reassurance look of CD4ζ CAR cells, an vital analytic affiliation of our after-effects is that the sufferers didn’t crave conditioning with lymphoablative regimens to perform abiding engraftment; lymphoablation has been all-important with arbor corpuscle therapies and for T corpuscle engraftment in blight sufferers. The assurance and abiding alternation of engineered T beef supplies a added account for cell-based HIV abolishment methods (38). Furthermore, these allegation accommodate a framework for the structure of abiding gene dedication methods for abiogenetic problems and added amiable altitude space chemotherapy will not be possible.

Each of the three CD4ζ trials had altered ameliorative protocols in antiretroviral biologic (ARV)–handled HIV topics. The Mitsuyasu abstraction (8) was a look 2 open-label trial, infusing a definite dosage of two × 1010 to three × 1010 CD4 and CD8 T beef per accountable with obvious HIV countless disconnected into two teams: (i) people who accustomed 6 × 106 U of IL-2 repeatedly alloyed over 4 canicule alpha 4 hours afore T corpuscle beverage or (ii) people who accustomed no IL-2. The Deeks abstraction (9) was a look 2 analytic balloon alleviative ARV-controlled HIV capability [levels abreast banned of apprehension (LODs)], and 1 × 1010 CD4/CD8 beef had been alloyed 3 times 2 weeks aside. Capacity had been enrolled assimilate a confederate of (i) these accepting CD4ζ-modified T beef or (ii) these accepting alone blunt T cells. The Aronson abstraction (clinicaltrials.gov NCT01013415) was a randomized, three-cohort balloon of capability with ARV-controlled HIV infections (ranges beneath detection). Two cohorts accustomed infusions of 8 × 109 to 9 × 109 CD4ζ-modified CD4/8 T beef with or afterwards subcutaneous injections of IL-2 (1.2 mIU/m2 per day) for 56 days. The third confederate accumulation accustomed IL-2 solely. For anniversary of those research, capability had been acutely monitored and evaluated afterwards beverage within the aboriginal yr for analytic responses, security, and allusive results. This deal with was initially undertaken as allotment of the FDA-mandated abiding aftereffect for ecology for delayed adversarial contest in sufferers accepting gene appraisal software amalgam vectors. Beneath this requirement, capability alternate in anniversary class and concrete exams as much as 15 years afterwards infusion. The accumulating of arctic PBMCs from the anniversary visits for anniversary accountable in anniversary of the research was compiled. The abyss of anniversary topic’s anniversary appointment aftereffect contour was primarily bent by the topic’s acquiescence with protocol-specified abstraction visits and to in a position case high quality.

A qPCR appraisal was developed to establish the amplicon fashioned by the CD4 and ζ chimera. gDNA for qPCR was deserted from arctic pellets of 1 × 106 PBMCs acquired from the buffy protecting of sweet claret from accountable anniversary visits with QIAamp DNA Micro Kit (Qiagen). qPCR was carried out in 20-μl volumes with the 384-well structure on ABI HT7900. Accurate CD4ζ and huGAPDH (human glyceraldehyde-3-phosphate dehydrogenase) primer/probe units had been acclimated to quantify corresponding archetype numbers of accountable samples from accepted curves. Appraisal after-effects had been evaluated by ABI’s SDS2.3, Excel, and SigmaPlot software program. To actuate the boilerplate CD4ζ copies/1 × 106 PBMCs, CD4ζ archetype numbers had been bent with 4 replicates of 250 ng of gDNA, and GAPDH archetype numbers had been bent with three replicates of fifty ng of gDNA and afresh acclimated to adapt the CD4ζ copies to 1 × 106 beef with the afterward formulation: 2 × (CD4ζ copies/GAPDH copies) × 1 × 106. TaqMan qPCR appraisal achievement archetype is as follows: absolute of altitude (LOQ) is about one archetype in 7600 cells, LOD is about one archetype in 38,000 cells, absolute ascendancy aural 80% of accepted worth, and R2 cost be better than 0.995.

A beeline mixed-effects archetypal (10) with the time again beverage as an absolute capricious was acclimated to appraisal the quantity of CD4ζ adulteration from yr 1 to yr 9 afterwards infusion. Application each unintentional intercepts and unintentional slopes, we modeled abeyant alternation amid afresh abstracts over time and between-subject airheadedness within the antecedent values, as able-bodied as the quantity of decay. For the accrued information, the aberration amid research within the yr 1 corpuscle counts was tailored by together with indicator variables of alone abstraction within the archetypal as covariates.

Cryopreserved accountable PBMCs had been aqueous and accustomed to steadiness transient afore absolute RNA was deserted with Qiagen RNeasy Plus equipment. The RNA was reverse-transcribed with alloyed hexamers and evaluated by TaqMan appraisal for the apprehension of CD4ζ and the advertence gene huGAPDH. A constant absolute Ct quantity for CD4ζ was afresh subtracted from the Ct quantity of the GAPDH to actuate ΔCt of CD4ζ about to GAPDH. As a ascendancy for CD4ζ DNA contamination, an RT acknowledgment afterwards the about-face transcriptase was executed for anniversary pattern. A abrogating arresting was start in all ascendancy samples, advertence that samples had no communicable DNA. The two samples with ephemeral CD4ζ RNA declared in Fig. 2A moreover had transgene archetype numbers beneath 100, suggesting that 0.01% of CD4ζ-modified T beef in PBMCs often is the absolute to establish CD4ζ RNA by this RT-TaqMan process.

Purified gDNA was digested with Mse I, Tsp 509I, or for Mu-mediated accretion (13), with Ban I. Samples with hooked up DNA quantities had been completed genome–amplified with the illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare) afore digestion. PCR adapters had been put in by T4 ligase or Mu transposase. Mse I and Tsp 509I samples had been afresh digested afresh with Msc I to anticipate accretion of agent sequence. Nested PCR was carried out with altitude declared forward (15) and primers particular to the CD4ζ agent LTR. Amplification articles had been antiseptic with AmPure chaplet and sequenced with 454 pyrophosphate sequencing know-how (39). Genomic sequences adjustment aural 3 bp of the LTR finish and assuming altered greatest alignments to the animal genome by BLAT (hg18, adaptation 36.1, >98% bout rating) had been suggested correct affiliation websites. Comparisons to genomic look had been carried out as declared forward (40, 41) with Fisher’s actual checks, χ2, or a mixture of codicillary logit, regression, and Bayesian archetypal averaging. An account of added genomic heatmaps may be start in Ocwieja et al. added argument “Guide to Interpreting Genomic Heat Maps Summarizing Affiliation Armpit Distributions” (42). Identification of cancer-related genes was executed with a accumulating of cancer-related gene lists from seven altered sources (http://microb230.med.upenn.edu/protocols/cancergenes.html). Affiliation armpit abstracts units acclimated and oligonucleotides acclimated for appraisal are listed in tables S5 and S6. The CD4ζ affiliation websites had been in comparison with affiliation websites recovered from added research that acclimated gammaretroviral vectors in T beef (17) and hematopoietic arbor beef (16).

www.sciencetranslationalmedicine.org/cgi/content material/full/4/132/132ra53/DC1

Fig. S1. Development and validation of the K562 activation appraisal for anatomic testing of CD4ζ cells.

Fig. S2. Affiliation patterns for CD4ζ are archetypal of gammaretroviral vectors.

Table S1. CD4ζ gene archetype cardinal and CD4ζ transcriptional motion in fixed cells.

Table S2. Affiliation websites recovered from CD4ζ topics.

Table S3. Affiliation websites recovered by >1 Mu Hop.

Table S4. Affiliation websites abreast array genes.

Table S5. Affiliation armpit abstracts units acclimated on this examine.

Table S6. Oligonucleotides acclimated on this examine.

G. Fitzmaurice, N. Laird, J. Ware, Applied Longitudinal Analysis. Wiley Series in Probability and Statistics (Wiley-Interscience, Hoboken, NJ, 2004).

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