Non-viral chimeric antigen receptor (CAR) T cells going viral.

Chimeric antigen receptor (CAR) T cell therapy has made significant strides in the treatment of B-cell malignancies, but its application in treating solid tumors still poses significant challenges. Particularly, the widespread use of viral vectors to deliver CAR transgenes into T cells comes with limitations, including high costs and regulatory restrictions, which hinder the translation of novel genetic engineering concepts into clinical applications. Non-viral methods, such as transposon/transposase and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems, offer promising alternatives for stable transgene insertion in CAR-T cells. These methods offer the potential to increase accessibility and efficiency in the development and delivery of CAR-T cell therapies. The main challenge in using non-viral methods, however, is their low knock-in efficiency, which leads to low transgene expression levels. In this review, we discuss recent developments in non-viral approaches for CAR-T cell production, the manufacturing requirements for clinical-grade production of non-viral CAR-T cells, and the adjustments needed in quality control for proper characterization of genomic features and evaluation of potential genotoxicity.

Human respiratory syncytial virus surface glycoproteins F, G and SH form an oligomeric complex.

In order to study structural associations, RSV surface glycoproteins were evaluated using heparin agarose affinity chromatography (HAAC). When RSV-infected cell lysate was analyzed by HAAC, all three surface glycoproteins, (F, G and SH), were eluted. Similarly, when separate lysates from Vero cells infected with vaccinia recombinants expressing F (vvF), G (vvG) and SH (vvSH) proteins were subjected to HAAC, only vvF and vvG expressed proteins bound to heparin, whereas vvSH expressed protein did not bind. When lysates from vvF, vvG and vvSH-infected Vero cells were mixed prior to HAAC, only F and G bound heparin. In contrast, following co-infection of Vero cells with vvF, vvG and vvSH, all three proteins were detected subsequent to HAAC. Following HAAC of A2-infected cell lysate and lysate from vvF, vvG and vvSH co-infected Vero cells, two high molecular weight complexes of 175 Kd and 210 Kd, respectively, were identified that reacted with anti-F, anti-G and anti-SH antisera. In addition, anti-SH antiserum was able to co-precipitate RSV F, G and SH. Using HAAC and a NaCl step gradient we demonstrated that a fraction of RSV F, G and SH eluted at higher salt concentrations than either purified F or G protein. Taken together, these data suggest that RSV F, G and SH glycoproteins can form an oligomeric complex within infected cells and this complex has a higher affinity for heparin than either G or F protein alone.

The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate.

Human respiratory syncytial virus (RSV) F glycoprotein (RSV-F) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. RSV-glycosaminoglycan (GAG) interactions were evaluated using heparin-agarose affinity chromatography. RSV-F from A2- and B1/cp-52 (cp-52)-infected cell lysates, RSV-F derived from a recombinant vaccinia virus, and affinity-purified F protein all bound to and were specifically eluted from heparin columns. In infectivity inhibition studies, soluble GAGs decreased the infectivity of RSV A2 and cp-52, with bovine lung heparin exhibiting the highest specific activity against both A2 (50% effective dose [ED(50)] = 0.28 +/- 0.11 microg/ml) and cp-52 (ED(50) = 0.55 +/- 0. 14 microg/ml). Furthermore, enzymatic digestion of cell surface GAGs by heparin lyase I and heparin lyase III but not chondroitinase ABC resulted in a significant reduction in cp-52 infectivity. Moreover, bovine lung heparin inhibited radiolabeled A2 and cp-52 virus binding up to 90%. Taken together, these data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity.

Identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein G.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (184A-->T198) and B (183K-->K197). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence 184A/183K-->198T/K197 for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.

The effect of rate of stimulation on force of contraction in a partially paralyzed rat phrenic nerve hemidiaphragm preparation.

This study was performed to determine whether presynaptic receptor blockade could be differentiated from postsynaptic blockade by examining the effect of increasing rates of indirect stimulation on twitch height depression (THD) on partially paralyzed in vitro rat diaphragm preparations. We calculated the T200/T1 ratio (force of the 200th stimuli divided by the force of the first stimuli) at rates of 0.2 Hz, 0.5 Hz, 1 Hz, and 2 Hz using a drug concentration which provided approximately 20% THD during stimulation at 0.1 Hz. Markedly different T200/T1 ratios were demonstrated when hexamethonium, a drug with predominantly presynaptic effects, was compared with alpha bungarotoxin, a drug with predominantly postsynaptic effects. These results were then compared with those from vecuronium, rocuronium, mivacurium, and tubocurarine. Both hexamethonium and rocuronium caused a marked decrease in T200/T1 ratio at higher rates of stimulation; alpha bungarotoxin caused a slight increase in T200/T1 ratio at higher rates of stimulation. The T200/T1 ratios produced by vecuronium, mivacurium, and tubocurarine lay intermediate between hexamethonium and alpha bungarotoxin. Significant differences in T200/T1 ratios were found when alpha bungarotoxin was compared with all other drugs at 2 Hz. Hexamethonium and rocuronium produced significant differences in T200/T1 ratio from those of all the other drugs at 1 Hz and 2 Hz. There were significant differences in the T200/T1 ratio found after hexamethonium and rocuronium compared to alpha bungarotoxin at 0.5 Hz. No significant differences at any rate of stimulation were found between hexamethonium and rocuronium. No difference was observed in the effect of vecuronium, mivacurium, and tubocurarine. We conclude that, if the observed effect is the result of hexamethonium acting predominantly at presynaptic sites and alpha bungarotoxin acting predominantly at postsynaptic sites, the relative contribution of small doses of nondepolarizing drugs at each site can be differentiated by determining the T200/T1 ratio at rates of 1 Hz or 2 Hz. Our results are consistent with the suggestion that small doses of rocuronium have marked presynaptic activity, but that vecuronium, mivacurium, and tubocurarine have both pre- and postsynaptic effects.

Tracheal intubation conditions after one minute: rocuronium and vecuronium, alone and in combination.

Rocuronium is a recently introduced nondepolarising neuromuscular blocking agent with a rapid onset and intermediate duration of action. Experimental observations have suggested that during onset it acts synergistically with other nondepolarising agents, but that at a steady state the combined action is additive. In order to investigate whether synergism during onset produces a clinical benefit we performed the following study of tracheal intubation conditions. Consenting patients presenting for elective surgery which required tracheal intubation were randomly allocated to receive a standard anaesthetic and either a twice ED95 dose of rocuronium, or vecuronium, or an equipotent mixture of both drugs. Tracheal intubation conditions were assessed after 60 s and scored as excellent, good, poor or impossible. The conditions produced in the rocuronium and the mixture groups were similar and both were significantly better than those of vecuronium. Excellent intubation conditions were achieved in 57% of the rocuronium group, 70% of the mixture group and 27% of the vecuronium group. We conclude that a mixture of rocuronium and vecuronium acts synergistically during the early part of their action and a mixture containing one ED95 of both agents provides comparable conditions for tracheal intubation as an equipotent dose of rocuronium.

Is rocuronium an exception to the relation between onset and offset? A comparison with pipecuronium.

A general relation between the rate of onset and rate of recovery from non-depolarizing blockade has been demonstrated, with recovery consistently about ten times slower than onset. This observation has led to the suggestion that non-depolarizing agents share a common mechanism of action. Rocuronium, a recently introduced steroidal non-depolarizing agent, is claimed to have a very rapid onset but an intermediate duration and appears to test this hypothesis. To investigate this paradox we have calculated the rates of onset and recovery of rocuronium using the isolated human forearm and compared them with those of pipecuronium. The mean ratio of recovery time/onset time for rocuronium was 31.3, which is significantly greater than that for pipecuronium, 11.6 (P < 0.01). Whilst pipecuronium conforms to the same general relation between onset and offset described previously for other non-depolarizing agents, rocuronium appears to have a disproportionately rapid rate of onset for its rate of recovery. This suggests that onset, recovery, or both onset and recovery, from rocuronium blockade occur in a different manner to that of other non-depolarizing agents.

The influence of cold on the recovery of three neuromuscular blocking agents in man.

The Arrhenius hypothesis suggests that change in temperature has a less marked effect on the rate of physical processes than on biological reactions. We have investigated the process underlying recovery from neuromuscular block in man by studying the effect of cooling on the rate of recovery from depolarising and non-depolarising block. Vecuronium, rocuronium and decamethonium (C10) neuromuscular block were investigated using the isolated forearm technique on awake human volunteers. In these experiments, one arm was cooled whilst the other was used as control. Moderate hypothermia decreased the rate of recovery from all three agents, but this was significantly less marked with the depolarising drug. The mean Q10 (the anticipated change in rate of a reaction across of 10 degrees C temperature gradient) of the rate of recovery for vecuronium was 3.21, rocuronium 2.86 and decamethonium 1.29. This suggests a different process in the recovery of these two types of drug. According to the Arrhenius hypothesis this would suggest that the recovery from non-depolarising drugs is likely to involve a biochemical mechanism and that recovery from decamethonium is controlled by a physical process.

Retrospective multicenter analysis of 3i endosseous dental implants placed over a five-year period.

Osseointegrated dental implants have now become an accepted form of treatment for patients with a fully or partially missing dentition. The purpose of this study was to evaluate the performance of 3i threaded and cylindrical implants. During a 5-year period, a total of 1969 3i endosseous implants were placed at 6 centers in the United States and 2 elsewhere in the edentulous and partially edentulous jaws of 653 consecutive patients ranging in age from 18 to 82 years. Of the total number of implants placed, 1341 were commercially pure titanium threaded implants and 628 were titanium plasma-sprayed implants with a cylindrical configuration. A total of 28 patients with 110 implants were lost to follow-up. Implants in these patients were considered neither a success nor a failure. Success was predicated on the osseointegration of each and every implant followed in this analysis rather than the persistence of prosthetic function. Confirmed bone anchorage was considered essential for success. A total of 625 patients with 1871 implants remain in the study with a follow-up period ranging from 6 to 60 months. A total of 93 implants (5.0% of the total implants followed) were considered as failures. A mean implant survival rate was 95.0% for both the threaded and the cylindrical implant was calculated. The success rate of threaded implants was 97.0% in the mandible and 93.8% in the maxilla. The success rate for the 3.3-mm cylindrical implants was 96.0% in the mandible and 95.5% in the maxilla, and the success rate of 4.0-mm-diameter cylindrical implants was 95% in the mandible and 92.0% in the maxilla. Causes of failure consisted of loss of osseointegration (2.3%), crestal bone loss requiring periodontal therapy after the first year of function (1.7%) had mechanical problems associated with the prosthesis (0.9%). This retrospective analysis of the 3i endosseous implant system is comparable to previous reports on other implant systems in terms of implant survival and prosthesis stability. It is demonstrated that 3i implants are predictable and can provide lasting osseointegration leading to improvement of oral function if the recommended surgical and restorative protocol is followed.

Interaction of decamethonium with hexamethonium or vecuronium in the rat: an isobolographic analysis.

We used isobolographic analysis to investigate the interaction of decamethonium with either hexamethonium or vecuronium in the rat phrenic nerve hemidiaphragm preparation. EC50 values of decamethonium, hexamethonium, and vecuronium were (mean +/- SEM) 47.36 +/- 9.58 microM, 4.27 +/- 0.53 mM, and 5.19 +/- 1.17 microM, respectively. Combinations of drugs in concentrations corresponding to the 1:2, 1:1, and 2:1 ratios of their EC50 values were used to determine three points of each isobole. Decamethonium and hexamethonium showed antagonism: significant deviations from the line of additivity were found at EC50 ratios of 2:1 and 1:1 (P < 0.01 and P < 0.05, respectively) indicating that hexamethonium is a potent antagonist of decamethonium. For decamethonium and vecuronium none of the three points on the isobole was significantly different from the corresponding point on the line of additivity. Hexamethonium is known to be a weak antagonist at the postsynaptic nicotinic acetylcholine receptor but a potent antagonist at the presynaptic nicotinic receptor. Vecuronium is a more potent antagonist at the postsynaptic nicotinic receptor but a much weaker antagonist at the presynaptic site. It was postulated that in the rat the primary site of action of decamethonium is at the presynaptic nerve terminal. Our findings suggest that presynaptic rather than postsynaptic potency of a nondepolarizing drug determines ability to antagonize the effect of a depolarizing drug in the rat.

Priming studies with rocuronium and vecuronium.

We studied the effects of rocuronium and vecuronium as priming agents before both vecuronium and rocuronium neuromuscular blockade. Rocuronium is ineffective at priming rocuronium. Vecuronium is effective at priming rocuronium, producing an approximate 33% reduction in onset time. Rocuronium and vecuronium, when given as priming agents, both reduce the onset time of vecuronium block.

The effect of dose and the rate of stimulation on the action of rocuronium.

We studied the effect of administering multiples of the ED90 dose of rocuronium on the onset, duration and recovery index of neuromuscular block. The effect on onset time differed from that reported using train-of-four stimulation. This led us to investigate the effect of increasing the rate of ulnar nerve stimulation 10-fold. This resulted in an approximately 50% reduction in the time to complete block.

No effect of circulating drug upon isolated forearm block.

Controversy exists as to whether the recovery of isolated arm blockade is primarily determined by resultant plasma drug concentrations, or by the affinity of the drug for the biophase. We have investigated the effect of the circulating drug produced by the isolated forearm experiment upon its recovery profile. Paralysis from retrograde spread of drug after the intravenous injection of 20 ml saline containing vecuronium 0.3 mg into a forearm isolated from the circulation was achieved in three groups of five experiments. Group 1 were used as controls, the tourniquet being released after 3-4 min and the recovery of block observed. In group 2 the tourniquet was similarly released but a repeat dose of vecuronium 0.3 mg was administered into the systemic circulation at 10% recovery. In group 3 the tourniquet was released at 50% twitch depression and the repeat dose of vecuronium 0.3 mg given when the twitch height had recovered to that level. The mean (SD) 25% to 75% recovery indices of groups 1, 2 and 3 were: 9.2 (2.4), 8.7 (1.2) and 9.9 (1.9) min. There was no noticeable effect on the recovery slope of any of the traces when the second dose of myoneural blocker was given systemically in groups 2 and 3. The findings indicate that the main determinant of recovery of the isolated forearm experiment is not its plasma drug concentration but a mechanism which maintains the drug in the effect compartment.

Rate of 'onset' and 'offset' of four non-depolarising neuromuscular blocking drugs.

There is evidence that onset time and potency of non-depolarising neuromuscular blocking drugs are related. The relationship between onset time and rate of recovery has not been investigated. In this study, using the isolated forearm technique, the onset time of four different non-depolarising drugs have been compared with their recovery times. It has been demonstrated that there is a strong relationship between these parameters. It is proposed that the relationship between onset time and offset time suggests a common mechanism that influences both events and that this may also explain the relationship between potency and the rate of onset.

Sensitivity to second dose of mivacurium.

The sensitivity of patients to a second dose of mivacurium has been studied following complete recovery of the twitch response after > 95% neuromuscular block produced by a systemic bolus of the drug. In further experiments we have excluded one arm from the effect of a systemic bolus ED95 dose of mivacurium for 100 s so as to obtain two different levels of neuromuscular block in the two arms of the same patient. Upon recovery from the block in the paralysed arm the dose response of both arms to a second dose of mivacurium was studied in order to investigate the effect of the amount and duration of block upon second dose sensitivity. An approximately 50% diminution in the ED95 dose requirement of mivacurium was found following complete recovery from an ED95 dose in spite of the rapid plasma clearance of this drug. A similar increase in sensitivity was observed in the arm that had been excluded for 100 s from the peak effect of the drug. It was concluded that the second dose sensitivity was not due to a receptor effect or to residual drug in plasma.

Curare modification of suxamethonium blockade.

Tubocurare (0.125 mg.kg-1 or 0.25 mg.kg-1) was injected 10 s before 1 mg.kg-1 suxamethonium in patients anaesthetised with enflurane 1-1.5%. Measurement of electromyographic response was recorded using a 0.2 Hz train-of-four every 20 s. The modified blocks were slower in onset, of lesser intensity, and of shorter duration than that of suxamethonium alone, but were much closer to those of suxamethonium than of tubocurare. However, the train-of-four fade observed during onset of the modified blocks were similar to that of their tubocurare controls and significantly different from the suxamethonium group. We conclude that effective amounts of tubocurare are present in the neuromuscular junction within the 30 s following intravenous injection of the drugs, and this affects the onset of action of the suxamethonium block. The presence of train-of-four fade during a predominantly agonist block is difficult to explain on the basis of diminished acetylcholine release and a postsynaptic site of action of suxamethonium.