An adenovirus vector with a chimeric fiber incorporating stabilized single chain antibody achieves targeted gene delivery.

Adenovirus (Ad) vectors are of utility for many therapeutic applications. Strategies have been developed to alter adenoviral tropism to achieve a cell-specific gene delivery capacity employing fiber modifications allowing genetic incorporation of targeting motifs. In this regard, single chain antibodies (scFv) represent potentially useful agents to achieve targeted gene transfer. However, the distinct biosynthetic pathways that scFv and Ad capsid proteins are normally routed through have thus far been problematic with respect to scFv incorporation into the Ad capsid. Utilization of stable scFv, which also maintain correct folding and thus functionality under intracellular reducing conditions, could overcome this restriction. We genetically incorporated a stable scFv into a de-knobbed, fibritin-foldon trimerized Ad fiber and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of cells. We have shown that the scFv employed in this study retains functionality and that stabilizing the targeting molecule, per se, is critical to allow retention of antigen recognition in the adenovirus capsid-incorporated context.

Antigen-independent selection of stable intracellular single-chain antibodies.

The intracellular expression of single-chain Fv antibody fragments (scFv) in eukaryotic cells has an enormous potential in functional genomics and therapeutics [Marasco (1997) Gene Ther. 4, 11-15; Richardson and Marasco (1995) Trends Biotechnol. 13, 306-310]. However, the application of these so-called intrabodies is currently limited by their unpredictable behavior under the reducing conditions encountered inside eukaryotic cells, which can affect their stability and solubility properties [Wörn et al. (2000) J. Biol. Chem. 275, 2795-2803; Biocca et al. (1995) Bio/Technology 13, 1110-1115]. We present a novel system that enables selection of stable and soluble intrabody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single-chain antibodies fused to a selectable marker that can control gene expression and cell growth. Our results show that the activity of a selectable marker fused to well characterized scFvs [Wörn et al. (2000) J. Biol. Chem. 275, 2795-2803] correlates with the solubility and stability of the scFv moieties. This method provides a unique tool to identify stable and soluble scFv frameworks, which subsequently serve as acceptor backbones to construct intrabody complementarity determining region libraries by randomization of hypervariable loops.

Conservation of glutamine-rich transactivation function between yeast and humans.

Several eukaryotic transcription factors such as Sp1 or Oct1 contain glutamine-rich domains that mediate transcriptional activation. In human cells, promoter-proximally bound glutamine-rich activation domains activate transcription poorly in the absence of acidic type activators bound at distal enhancers, but synergistically stimulate transcription with these remote activators. Glutamine-rich activation domains were previously reported to also function in the fission yeast Schizosaccharomyces pombe but not in the budding yeast Saccharomyces cerevisiae, suggesting that budding yeast lacks this pathway of transcriptional activation. The strong interaction of an Sp1 glutamine-rich domain with the general transcription factor TAF(II)110 (TAF(II)130), and the absence of any obvious TAF(II)110 homologue in the budding yeast genome, seemed to confirm this notion. We reinvestigated the phenomenon by reconstituting in the budding yeast an enhancer-promoter architecture that is prevalent in higher eukaryotes but less common in yeast. Under these conditions, we observed that glutamine-rich activation domains derived from both mammalian and yeast transcription factors activated only poorly on their own but strongly synergized with acidic activators bound at the remote enhancer position. The level of activation by the glutamine-rich activation domains of Sp1 and Oct1 in combination with a remote enhancer was similar in yeast and human cells. We also found that mutations in a glutamine-rich domain had similar phenotypes in budding yeast and human cells. Our results show that glutamine-rich activation domains behave very similarly in yeast and mammals and that their activity in budding yeast does not depend on the presence of a TAF(II)110 homologue.

Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors.

A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of beta-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.

A trans-acting peptide activates the yeast a1 repressor by raising its DNA-binding affinity.

The cooperative binding of gene regulatory proteins to DNA is a common feature of transcriptional control in both prokaryotes and eukaryotes. It is generally viewed as a simple energy coupling, through protein-protein interactions, of two or more DNA-binding proteins. In this paper, we show that the simple view does not account for the cooperative DNA binding of a1 and alpha2, two homeodomain proteins from budding yeast. Rather, we show through the use of chimeric proteins and synthetic peptides that, upon heterodimerization, alpha2 instructs a1 to bind DNA. This change is induced by contact with a peptide contributed by alpha2, and this contact converts a1 from a weak to a strong DNA-binding protein. This explains, in part, how high DNA-binding specificity is achieved only when the two gene regulatory proteins conjoin. We also provide evidence that features of the a1-alpha2 interaction can serve as a model for other examples of protein-protein interactions, including that between the herpes virus transcriptional activator VP16 and the mammalian homeodomain-containing protein Oct-l.

Gene activation at a distance and telomeric silencing are not affected by yeast histone H1.

Until recently, it was believed that the budding yeast Saccharomyces cerevisiae has no histone H1 gene. However, a search of the yeast genome database revealed a possible H1 homologue of 258 amino acids, termed yeast histone H1 (HHO1). The protein shows 36% identity to the human H1 core domain over a stretch of 93 amino acids. Unlike other H1 proteins, Hho1p has a second possible core domain which shows 43% identity to the first core domain. Since vertebrate H1 histone had been implied in gene repression as well as gene activation at a distance, we tested the effect of deleting the yeast H1-like gene on remote activation of a modified GAL1 promoter, which contains a synthetic GAL4 binding site close to the TATA box, and the natural UASG, consisting of four GAL4 binding sites. Different spacing up to 1.8 kb between the proximal binding site and the distal UASG enhancer revealed no differences in gene activation between wild-type and knockout strains. Overexpression of a heterologous histone H1 from sea urchin showed an overall inhibition of gene activation by the GAL1 promoter, whereas overexpression of the yeast histone H1 had no effect. Also, the expression of A1, ALPHA2 or SUC2 genes, all of which are known to be responsive to an altered chromatin structure, was unchanged in HHO1 knockout or HHO1-overexpressing strains when compared to wild-type cells. We also considered the possibility that HHO1 was involved in forming the heterochromatin at telomeres. On testing for telomeric silencing of a URA reporter gene introduced 1.3 kb away from the chromosomal end, we again observed no differences between wild-type and knockout strains. Thus, the yeast histone H1-like gene appears to have no role in gene activation at a distance or in silencing under the conditions tested. It remains to be seen whether the yeast H1 histone is a gene-specific regulator rather than a general chromatin-associated protein.

Improved "activator trap" method for the isolation of transcriptional activation domains from random DNA fragments.

We have previously developed an "activator trap" method for selective isolation of activation domains from viral and cellular transcription factors. In this method, random sonicated DNA fragments were ligated next to the DNA binding domain (DBD) of the GAL4 factor in a plasmid that also contained a simian virus 40 (SV40) replication origin. A library of such random insert plasmids was transfected into a monkey cell line (CV-1-5GT), which had been stably transformed with a GAL4-inducible SV40 T-antigen gene. Chimeric GAL factors with a heterologous activation domain were harvested after selective replication in these CV-1-5GT cells. Here we report a simplification and generalization of the "activator trap" method. First, the time-consuming library construction step can be omitted by direct transfection of the sonicated DNA fragments and the linearized recipient plasmid vector into CV-1-5GT cells to obtain chimeric GAL-activation factors by in vivo ligations. Second, the dependence on CV-1-5GT cells can be bypassed by direct co-transfection of all components, including a plasmid carrying the T-antigen gene into cells other than CV-1-5GT. This latter step allows the application of the method to cultured human cells, as demonstrated with the human B-cell line BJA-B.

The cardiointegram: detection of coronary artery disease in males with chest pain and a normal resting electrocardiogram.

The cardiointegram is a non-invasive technique for the analysis of the electrical signals of the heart obtained by a transformation of the voltage vs. time format by a series of integrations. This multicenter study compares the results of the cardiointegram with coronary arteriography in 140 male patients with chest pain and a normal resting electrocardiogram. The cardiointegram was determined on two resting complexes of Leads I, II, V4, V5 and V6 and called abnormal if greater than or equal to four of ten complexes were abnormal, i.e., fell outside of a previously determined template of normality. The sensitivity was 73% and specificity was 78% for the diagnosis of occlusive coronary artery disease. When greater than or equal to five of ten abnormal complexes were used as the cut-off for an abnormal test and "equivocal" results (four of ten abnormal, n = 18) were excluded from analysis there was a sensitivity of 69% and specificity of 88%. Thirty-seven of 38 patients (97%) with an abnormal cardiointegram and a positive exercise stress test had coronary artery disease. Thus, the cardiointegram appears to be a useful non-invasive test for the detection of coronary artery disease in males with chest pain and a normal resting electrocardiogram in whom the diagnosis of coronary artery disease is being considered.

Pacemakers of the 1980s.

Today's pacing techniques and technologies have gone a long way toward the ideal and in the 1980s will make further strides toward a possible universally responsive, sophisticated, single instrument. These advances include the use of programmability of at least mode, rate, output, sensitivity, and refractory period; dual-chamber as well as single-chamber pacing; CMOS and LSI circuits; lithium batteries and possibly a limited revival of plutonium batteries; new lighter, longer-lasting, positive attachment leads; and new methods of triggering pacing. However, the increase in complexity and expense of the new devices has led to a question of their need, to a disputed question as to the possibility of overuse of pacing in general, and a need to reconcile government-mandated cost containment with medical as well as socioeconomic reality.

Early malfunction of transvenous pacemaker electrodes. A three-center study.

A 3-year study by three medical centers has revealed a 1-year electrode malfunction rate of 7.4%; most malfunctions occurred within the first 30 days. The incidence of unavoidable early malfunction (3.2%) fell within the 5% standards suggested by the committee report of the Inter-Society Committee on Heart Diseases. Incidences of obscure cause (3.2%) may be difficult to identify prospectively and may be, to a certain extent, unavoidable. The majority of the malfunctions (4.2%) showed specific clues that indicated that they were preventable. Successful repositioning was achieved on the first attempt in 80.6% of the cases with malfunction, and only 0.7% required ultimate myocardial electrode implantation. The principal clues to potentially unsatisfactory positioning included the presence of a large right ventricle with or without tricuspid insufficiency, current thresholds greater than 0.5 mA and ST-segment deviations on the intracardiac electrogram of less than 2 mV. Electrode malfunction may be more common with bipolar than with unipolar electrodes; but significant differences in the incidence of malfunction among different unipolar electrodes were observed. These data indicate that further developments in transvenous electrode design are warranted.

Cardiac pacing and pacemakers II. Serial electrophysiologic-pharmacologic testing for control of recurrent tachyarrhythmias.

The place of pacemakers in the treatment of tachyarrhythmias has expanded far beyond the initial role in the brady-tachy syndrome, of providing a "minimum guaranteed rate" while medications suppress the tachycardia. Techniques have been developed for prevention, termination, and duplication of a patient's spontaneous tachycardia under safe catheterization laboratory conditions. Combined with accumulating information about the normal responses to electrophysiologic stresses, these techniques have led to a new dimension in arrhythmia control. Most tachycardias previously felt to be refractory can be controlled after serial electrophysiologic-pharmacologic testing, during which sequential pharmacologic and pacer regimens are tested until a combination is found which prevents induction of tachycardias, and/or a pace mode is found which reliably terminates the tachycardia. Use of such an approach reduces hospital admissions and referral for surgery, and eliminates prolonged hospitalization for assessment of therapy in patients with infrequent but potentially lethal spontaneous tachycardias.

Pacemaker failures characterized by continuous direct current leakage.

Pulse generator failure caused by continuous leakage of direct current through an output capacitor has not previously been appreciated. Routine post-explant electronic evaluation has identified the defect in six implanted and one external pulse generator. The constant direct current in the implantable units, 0.14 to 0.26 milliamperes, is in the range that produces ventricular arrhythmias in dogs although this did not occur in our patients. Evidence of local myocardial damage existed in four cases and of electrode deterioration in three. The implant failures occurred without warning and in four cases within 2 weeks of demonstrated normal function, blunting the predictive benefits of pacemaker monitoring programs. Capacitor discharge circuits used in many pacers are inherently capable of developing direct current leakage in the event of output capacitor short circuit. In one model of pacemakers such continuous direct current leakage caused 8.3 percent (3 of 36) of pulse generator failures, widely scattered in time at 23, 27 and 46 months after implant. Capacitor short circuit causing constant direct current leakage can masquerade as primary battery failure and should be suspected when cessation of pacer function is associated with increased threshold or poor myocardial electrogram without evidence of wire break or displacement.

Transtelephone pacemaker monitoring: five years later.

Six hundred nineteen patients have been followed by remote monitoring of pacemaker function using ECG and rate or rate alone; 278 of 280 have had battery exhaustion or electronic failure demonstrated. Ten percent of exhausted pacemakers failed prior to the average longevity of the particular model, and 32% (89 of 280) exceeded 36 months' longevity; of these, 13% (37 of 280) lasted more than 40 months and 4.6% (13 of 280) exceeded 50 months. The error rate is 0.7% (2 of 280). With pulse generator longevity increasing, monitoring is done less frequently during the first 2 years, then calls are made weekly after 24 months.