Anti-BCMA CAR T administration in a relapsed and refractory multiple myeloma patient after COVID-19 infection: a case report.

BACKGROUND: Very little is known about the risk that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection poses to cancer patients, many of whom are immune compromised causing them to be more susceptible to a host of infections. As a precautionary measure, many clinical studies halted enrollment during the initial surge of the global Novel Coronavirus Disease (COVID-19) pandemic. In this case report, we detail the successful treatment of a relapsed and refractory multiple myeloma (MM) patient treated with an anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cell therapy immediately following clinical recovery from COVID-19. CASE PRESENTATION: The 57 year old Caucasian male patient had a 4-year history of MM and was considered penta-refractory upon presentation for CAR T cell therapy. He had a history of immunosuppression and received one dose of lymphodepleting chemotherapy (LDC) the day prior to COVID-19 diagnosis; this patient was able to mount a substantial immune response against the SARS-CoV-2 virus, and antiviral antibodies remain detectable 2 months after receiving anti-BCMA CAR T cell therapy. The recent SARS-CoV-2 infection in this patient did not exacerbate CAR T-associated cytokine release syndrome (CRS) and conversely the CAR T cell therapy did not result in COVID-19-related complications. One month after CAR T cell infusion, the patient was assessed to have an unconfirmed partial response per International Myeloma Working Group (IMWG) criteria. CONCLUSION: Our case adds important context around treatment choice for MM patients in the era of COVID-19 and whether CAR T therapy can be administered to patients who have recovered from COVID-19. As the COVID-19 global pandemic continues, the decision of whether to proceed with CAR T cell therapy will require extensive discussion weighing the potential risks and benefits of therapy. This case suggests that it is possible to successfully complete anti-BCMA CAR T cell therapy after recovery from COVID-19. CRB-402 study registered 6 September 2017 at clinicaltrials.gov (NCT03274219).

International neurocognitive normative study: neurocognitive comparison data in diverse resource-limited settings: AIDS Clinical Trials Group A5271.

Infrastructure for conducting neurological research in resource-limited settings (RLS) is limited. The lack of neurological and neuropsychological (NP) assessment and normative data needed for clinical interpretation impedes research and clinical care. Here, we report on ACTG 5271, which provided neurological training of clinical site personnel and collected neurocognitive normative comparison data in diverse settings. At ten sites in seven RLS countries, we provided training for NP assessments. We collected normative comparison data on HIV- participants from Brazil (n = 240), India (n = 480), Malawi (n = 481), Peru (n = 239), South Africa (480), Thailand (n = 240), and Zimbabwe (n = 240). Participants had a negative HIV test within 30 days before standardized NP exams were administered at baseline and 770 at 6 months. Participants were enrolled in eight strata, gender (female and male), education (<10 and ≥10 years), and age (<35 and ≥35 years). Of 2400 enrolled, 770 completed the 6-month follow-up. As expected, significant between-country differences were evident in all the neurocognitive test scores (p < 0.0001). There was variation between the age, gender, and education strata on the neurocognitive tests. Age and education were important variables for all tests; older participants had poorer performance, and those with higher education had better performance. Women had better performance on verbal learning/memory and speed of processing tests, while men performed better on motor tests. This study provides the necessary neurocognitive normative data needed to build infrastructure for future neurological and neurocognitive studies in diverse RLS. These normative data are a much-needed resource for both clinicians and researchers.

Renal and metabolic toxicities following initiation of HIV-1 treatment regimen in a diverse, multinational setting: a focused safety analysis of ACTG PEARLS (A5175).

BACKGROUND: Convenient dosing, potency, and low toxicity support use of tenofovir disoproxil fumarate (TDF) as preferred nucleotide reverse transcriptase inhibitor (NRTI) for HIV-1 treatment. However, renal and metabolic safety of TDF compared to other NRTIs has not been well described in resource-limited settings. METHODS: This was a secondary analysis examining the occurrence of renal abnormalities (RAs) and renal and metabolic serious non-AIDS-defining events (SNADEs) through study follow-up between participants randomized to zidovudine (ZDV)/lamivudine/ efavirenz and TDF/emtricitabine/efavirenz treatment arms within A5175/PEARLS trial. Exact logistic regression explored associations between baseline covariates and RAs. Response profile longitudinal analysis compared creatinine clearance (CrCl) over time between NRTI groups. RESULTS: Twenty-one of 1,045 participants developed RAs through 192 weeks follow-up; there were 15 out of 21 in the TDF arm (P = .08). Age 41 years or older (odds ratio [OR], 3.35; 95% CI, 1.1-13.1), his- tory of diabetes (OR, 10.7; 95% CI, 2.1-55), and lower baseline CrCl (OR, 3.1 per 25 mL/min decline; 95% CI, 1.7-5.8) were associated with development of RAs. Renal SNADEs occurred in 42 participants; 33 were urinary tract infections and 4 were renal failure/insufficiency; one event was attributed to TDF. Significantly lower CrCl values were maintained among patients receiving TDF compared to ZDV (repeated measures analysis, P = .05), however worsening CrCl from baseline was not observed with TDF exposure over time. Metabolic SNADEs were rare, but were higher in the ZDV arm (20 vs 3; P < .001). CONCLUSIONS: TDF is associated with lower serious metabolic toxicities but not higher risk of RAs, serious renal events, or worsening CrCl over time compared to ZDV in this randomized multinational study.

Predictors of CNS injury as measured by proton magnetic resonance spectroscopy in the setting of chronic HIV infection and CART.

The reasons for persistent brain dysfunction in chronically HIV-infected persons on stable combined antiretroviral therapies (CART) remain unclear. Host and viral factors along with their interactions were examined in 260 HIV-infected subjects who underwent magnetic resonance spectroscopy (MRS). Metabolite concentrations (NAA/Cr, Cho/Cr, MI/Cr, and Glx/Cr) were measured in the basal ganglia, the frontal white matter, and gray matter, and the best predictive models were selected using a bootstrap-enhanced Akaike information criterion (AIC). Depending on the metabolite and brain region, age, race, HIV RNA concentration, ADC stage, duration of HIV infection, nadir CD4, and/or their interactions were predictive of metabolite concentrations, particularly the basal ganglia NAA/Cr and the mid-frontal NAA/Cr and Glx/Cr, whereas current CD4 and the CPE index rarely or did not predict these changes. These results show for the first time that host and viral factors related to both current and past HIV status contribute to persisting cerebral metabolite abnormalities and provide a framework for further understanding neurological injury in the setting of chronic and stable disease.

Improved neuropsychological and neurological functioning across three antiretroviral regimens in diverse resource-limited settings: AIDS Clinical Trials Group study a5199, the International Neurological Study.

BACKGROUND: AIDS Clinical Trials Group (ACTG) A5199 compared the neurological and neuropsychological (NP) effects of 3 antiretroviral regimens in participants infected with human immunodeficiency virus type 1 (HIV-1) in resource-limited settings. METHODS: Participants from Brazil, India, Malawi, Peru, South Africa, Thailand, and Zimbabwe were randomized to 3 antiretroviral treatment arms: A (lamivudine-zidovudine plus efavirenz, n = 289), B (atazanavir, emtricitabine, and didanosine-EC, n = 293), and C (emtricitabine-tenofovir-disoproxil fumarate plus efavirenz, n = 278) as part of the ACTG PEARLS study (A5175). Standardized neurological and neuropsychological (NP) screening examinations (grooved pegboard, timed gait, semantic verbal fluency, and finger tapping) were administered every 24 weeks from February 2006 to May 2010. Associations with neurological and neuropsychological function were estimated from linear and logistic regression models using generalized estimating equations. RESULTS: The median weeks on study was 168 (Q1 = 96, Q3 = 192) for the 860 participants. NP test scores improved (P < .05) with the exception of semantic verbal fluency. No differences in neurological and neuropsychological functioning between treatment regimens were detected (P > .10). Significant country effects were noted on all NP tests and neurological outcomes (P < .01). CONCLUSIONS: The study detected no significant differences in neuropsychological and neurological outcomes between randomized ART regimens. Significant improvement occurred in neurocognitive and neurological functioning over time after initiation of ARTs. The etiology of these improvements is likely multifactorial, reflecting reduced central nervous system HIV infection, better general health, and practice effects. This study suggests that treatment with either of the World Health Organization -recommended first-line antiretroviral regimens in resource-limited settings will improve neuropsychological functioning and reduce neurological dysfunction. CLINICAL TRIALS REGISTRATION: NCT00096824.

Pooled week 96 results of the phase III DUET-1 and DUET-2 trials of etravirine: further analysis of adverse events and laboratory abnormalities of special interest.

OBJECTIVES: The aim of the study was to investigate the frequency and severity of adverse events (AEs) and laboratory abnormalities of interest over 96 weeks of treatment with etravirine or placebo in the pooled TMC125 DUET (Demonstrate Undetectable viral load in patients Experienced with ARV Therapy) trials. METHODS: Treatment-experienced, HIV-1-infected patients randomly received etravirine 200 mg twice a day (bid) or placebo, plus a background regimen. The frequency and severity of neuropsychiatric, rash, hepatic and lipid AEs were analysed; frequencies were also adjusted for total patient-years of exposure (PYE). RESULTS: A total of 599 and 604 patients received etravirine and placebo, respectively (median treatment duration 96.0 and 69.6 weeks, respectively). There was no significant difference between the treatment groups in the frequency of neuropsychiatric AEs. However, a significant difference in the frequency of rash was observed (20.5% vs. 11.8%, respectively; P < 0.0001); rash was generally mild to moderate in severity; the rate of discontinuation because of rash was low (2.2% vs. 0% in the etravirine and placebo groups, respectively). The frequency of hepatic AEs was low and similar between the treatment groups (8.7% vs. 7.1%, respectively; P = 0.3370); hepatic enzyme levels did not increase over time. Lipid-related laboratory abnormalities and changes over time in lipid levels were generally comparable between treatment groups. Adjusting for treatment exposure, the frequency of AEs remained similar between treatment groups, with the exception of rash [13.7 vs. 9.3 per 100 PYE; relative risk (95% confidence interval) 1.48 (1.02-1.95)]. CONCLUSIONS: The frequency of AEs of interest was generally similar between the treatment groups, both overall and when adjusted for treatment exposure, with the exception of rash which was more frequent in the etravirine group.

Primary pulmonary hypertension, Castleman's disease and human herpesvirus-8.

Primary pulmonary hypertension (PPH) and Castleman's disease (CD) are rare conditions infrequently encountered in clinical practice. In this paper, two patients diagnosed with both of these diseases are reported. The authors speculate that rather than being a chance occurrence, these conditions are linked by a common angio-proliferative mechanism. Therefore, an association between infection with the human herpesvirus-8 and the diseases of PPH and CD was sought. Evidence of human herpesvirus-8 infection was found in the lung tissue and, specifically, in the plexiform lesions from one of the patients.

Relationship between human immunodeficiency virus type 1 (HIV-1)-specific memory cytotoxic T lymphocytes and virus load after recent HIV-1 seroconversion.

Human immunodeficiency virus type 1 (HIV-1)-specific memory, or precursor, cytotoxic T lymphocytes (CTL) in 14 subjects who had recently experienced seroconversion were evaluated with respect to virus set point, defined as plasma HIV-1 RNA level 6 months after seroconversion. Env-, Gag-, Pol-, and Nef-specific precursor CTL were detected in (51)Cr-release assays, using antigen-stimulated peripheral blood mononuclear cells as effectors and B cell lines infected with HIV-1-vaccinia recombinants as targets. All subjects tested had precursor CTL specific to at least 2 HIV-1 antigens. Detection of Env-specific precursor CTL was associated with a high set point (P=.0221). The number of antigens recognized tended to be greater in subjects with higher set points (rho=.45621; P=.1171). Gag-specific precursor CTL frequency correlated inversely with set point (rho=-.8478; P=.0003). Two heterozygotes for a 32-bp deletion in CCR5 had the lowest set points (P=.0220) and highest Gag precursor CTL frequencies (P=.0128). These data suggest that host factors that restrict viral replication may be important determinants of the level of HIV-1-specific precursor CTL.

Relationship of human herpesvirus 8 peripheral blood virus load and Kaposi's sarcoma clinical stage.

OBJECTIVE: To determine the relationship between human herpesvirus 8 (HHV-8 or Kaposi's sarcoma-associated herpesvirus) peripheral blood virus load and Kaposi's sarcoma (KS) clinical stage. DESIGN: Blinded, cross-sectional analysis of peripheral blood HHV-8 DNA levels in persons with AIDS-related KS in Harare, Zimbabwe. METHODS: Subjects were stratified by KS clinical stage. The amount of HHV-8 DNA in plasma and peripheral blood mononuclear cells (PBMC) was determined by quantitative real-time PCR amplification of the HHV-8 open reading frame 26. RESULTS: Thirty-one HIV-1/HHV-8-coinfected persons were studied: 26 subjects had histologically confirmed KS (one stage II, 11 stage III and 14 stage IV) and five subjects had antibodies to HHV-8 but did not have KS. The age, CD4 lymphocyte count and plasma HIV-1 RNA levels were similar in all groups. HHV-8 DNA was detected in the plasma of all HHV-8-infected subjects (range < 2.4 to 5.2 log10 copies/ml), but plasma HHV-8 DNA levels were not associated with KS disease stage. In contrast, the amount of HHV-8 DNA in PBMC (range < 0.7 to 4.5 log10 copies/microg) was strongly associated with KS clinical stage (P = 0.005). Among stage IV KS cases, there was a linear relationship between plasma and PBMC HHV-8 DNA levels (r2 = 0.42; P = 0.01). CONCLUSION: The strong association observed between the extent of KS disease and the levels of HHV-8 DNA in PBMC provides further evidence for a relationship between HHV-8 virus load and KS pathogenesis.

Quantitation of cell-free and cell-associated Kaposi's sarcoma-associated herpesvirus DNA by real-time PCR.

A real-time PCR assay for quantitation of Kaposi's sarcoma-associated herpes virus (KSHV or human herpesvirus 8) DNA was evaluated. The linear dynamic range was 10 to 10(5) copies of KSHV DNA (r(2) > 0.99). The accuracy of DNA purification and quantitation was less than +/-0.4 log(10) copies for samples that contained from 10 to 10(5) copies of KSHV DNA. Cell-associated KSHV DNA was quantitated over a range of infected cell frequencies from 0. 1 to 10(-5), and cell-free KSHV DNA in plasma was quantitated over a range of 100 to 10(6) copies/ml. Real-time PCR provides a convenient method for quantitation of cell-free and cell-associated KSHV DNA in laboratory and clinical specimens.

Changes in human immunodeficiency virus type 1 virus load during mobilization and harvesting of hemopoietic progenitor cells. Adult AIDS Clinical Trials Group 285 Study Team.

Genetic modification of hemopoietic progenitor cells ex vivo, followed by the infusion of the genetically modified cells into the human immunodeficiency virus-1 (HIV-1) infected donor, has been proposed as a treatment for HIV-1 infection. The current study was undertaken to evaluate the effect of hemopoietic stem cell mobilization and harvesting on HIV-1 replication in persons with HIV-1 infection. Eighteen HIV-1-infected persons received recombinant granulocyte colony-stimulating factor (G-CSF; Filgrastim) 10 microg/kg per day, for 7 days. On days 4 and 5, peripheral blood mononuclear cells were harvested by leukapheresis. The CD4+ lymphocyte count at entry was >500/microL for 6 subjects, 200 to 500/microL for 6 subjects, and <200/microL for 6 subjects. For 9 of 18 subjects, plasma HIV-1 RNA levels increased 4- to 100-fold (>0.6 log(10)) above baseline between days 4 and 7 and returned to baseline by day 27. Significant increases of plasma HIV-1 RNA levels occurred in 5 subjects despite 3-drug antiretroviral therapy. Changes in CD4+ and CD34+ cells during mobilization and harvesting were similar in all subjects whether they had or did not have increased plasma HIV-1 RNA levels. Thus, mobilization and harvesting of bone marrow progenitor cells from persons infected with HIV-1 induced a transient increase in viral replication in some patients but was not associated with adverse effects. (Blood. 2000;95: 48-55)

Reduced mobilization of CD34+ stem cells in advanced human immunodeficiency virus type 1 disease.

Granulocyte colony-stimulating factor (r-met Hu G-CSF; filgrastim; 10 microgram/kg/day for 7 days) was used to mobilize CD34+stem cells into the peripheral blood of human immunodeficiency virus type 1 (HIV-1)-infected individuals and a group of HIV-1-uninfected donors as a measure of immunologic reserve in HIV-1-infected people. G-CSF mobilized CD34+ cells of HIV-1-infected individuals with cell counts >500 CD4+ cells/mm3, as well as in HIV-1-uninfected donors. In contrast, CD34 cell mobilization was significantly blunted in HIV-1-infected individuals with cell counts <500 CD4+ cells/mm3 (<200 cell days vs. >650 cell days, P<.0005, compared with the >500 CD4+ cell cohort). At least 1.75x10(7) CD34 cells were harvested by leukapheresis from patients in each study cohort. CD34+ cell viability and the ability to differentiate precursor cells into myeloid and erythroid progenitor cells were not affected by HIV-1 infection.

Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) infection in men receiving treatment for HIV-1 infection.

OBJECTIVE: To determine the prevalence of human herpesvirus 8 (HHV-8) infection in men treated for HIV-1 infection in Denver, Colorado. DESIGN: Cross-sectional analysis METHODS: Blood samples were obtained from 216 HIV-1-infected men. Antibody to latency-associated nuclear antigen (LANA) was detected by an immunofluorescent assay and the presence of HHV-8 in peripheral blood mononuclear cells (PBMC) was detected by polymerase chain reaction amplification. RESULTS: Among HIV-1-infected men who did not have Kaposi's sarcoma (KS), prevalence of HHV-8 infection was 46% (95% confidence interval [CI], 0.39-0.52). LANA seropositivity was common both among subjects with KS and subjects without KS (69% versus 42%; p = .06), but detection of HHV-8 DNA in peripheral blood was strongly associated with a diagnosis of KS (44% versus 10%; p = .001). In a univariate analysis of study subjects without KS, neither the odds of LANA seropositivity nor detection of HHV-8 DNA in PBMC was significant for CD4+ lymphocyte count, HIV-1 virus load, the use of three drug antiretroviral regimens or the prior occurrence of non-KS AIDS-related conditions. CONCLUSION: Although antibodies to HHV-8 are common among HIV-1-infected men, detection of HHV-8 DNA in PBMC is uncommon and is associated with a diagnosis of Kaposi's sarcoma.

Comparison of the prevalence of antibodies to human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) in Brazil and Colorado.

The prevalence of human herpesvirus 8 (HHV-8; Kaposi's sarcoma [KS] herpesvirus) infection was determined by IFA in 297 persons living in Brazil and Colorado. The prevalence of antibody to HHV-8 in human immunodeficiency virus (HIV) type 1-seropositive gay men with and without KS was similar in Brazil and Colorado. In Brazil, the prevalence of HHV-8 antibody was significantly greater in HIV-1-seronegative gay men than in HIV-1-seronegative male intravenous drug users. HHV-8-seropositive Brazilian gay men who had a clinical diagnosis of KS or who were infected with HIV-1 had significantly higher titers of HHV-8 antibody than did HHV-8-seropositive, HIV-1-seronegative Brazilian gay men. These findings provide further support for the association between HHV-8 infection and KS and suggest that, as in the United States, HHV-8 infection is transmitted sexually in Brazil.

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.

Mutations in the Tetrahymena ribozyme internal guide sequence: effects on docking of the P1 helix into the catalytic core and correlation with catalytic activity.

Binding of substrate by the ribozyme derived from the self-splicing intron of Tetrahymena thermophila involves at least two steps. In the first step, base pairing between the ribozyme internal guide sequence (IGS) and the substrate forms a helical duplex (P1). Through specific tertiary interactions between P1 and the ribozyme core, P1 is then docked into the ribozyme active site. We have investigated the effects of compensatory mutations in positions 2-6 of the P1 helix on docking of P1 into the ribozyme core. Equilibrium binding of matching oligonucleotides by catalytically active IGS mutant ribozymes was evaluated by gel-shift analysis. While the strength of base pairing changed with base composition as expected, the strength of tertiary interactions between P1 and the ribozyme core was not affected by the P1 mutations. These results support a model in which efficient docking of P1 is determined by P1 structure and the presence of a conserved G-U pair. Determination of the rate of dissociation of matching oligonucleotides from each ribozyme revealed that mutations in the IGS change the tightness of binding by increasing or decreasing the dissociation rate. Surprisingly, dissociation rates determined in this fashion were 20-900-fold less than the values of the multiple-turnover rate constant for these ribozymes, initially suggesting that turnover did not require product dissociation. A more detailed analysis for the wild-type ribozyme defined two distinct product dissociation rates. The slower rate equaled that determined under the conditions used for the equilibrium binding studies. The weighted average of the two dissociation rates equaled the multiple-turnover rate constant. These results are explained by a model in which ribozyme preparations consist of two ribozyme conformers: one with tight docking of P1 and another with weaker docking of P1.

Phase 1 study of combination therapy with L-697,661 and zidovudine. The ACTG 184 Protocol Team.

We performed a pilot study that examined the clinical and pharmacokinetic interactions between zidovudine (ZDV) and a pyridinone derivative, L-697-661. The results indicate that the drugs were well tolerated, with no important pharmacokinetic interactions, when administered concomitantly for as long as 8 weeks. Although the number of study participants was small, we noted rapid emergence of resistance to L-697,661 among ZDV-naive study subjects who were administered L-697,661 as monotherapy but did not observe isolates of human immunodeficiency virus type 1 (HIV-1) resistant to L-697,661 among those who were administered concomitant ZDV. These results suggest a potential interaction between development of resistance to L-697,661 and ZDV. Although the clinical development of L-697,661 has been halted, our results support the need for further studies to test whether specific interactions among antiretroviral agents administered in combination and the molecular target can delay the emergence of isolates that exhibit resistance to all drugs in the regimen.

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library.