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The nonclinical safety assessments for gene therapies are evolving, leveraging over 20 years of experimental and clinical experience. Despite the growing experience with these therapeutics, there are no approved harmonized global regulatory documents for developing gene therapies with only the ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) S12 guidance on nonclinical biodistribution currently under discussion. Several health authorities have issued guidance over the last 15 years on the nonclinical safety aspects for gene therapy products, but many of the recommendations are limited to high-level concepts on nonclinical safety aspects or altogether silent on key topics. Historically, this approach was appropriately vague given our relatively small dataset of nonclinical experience, where a comprehensive and detailed regulatory guidance approach was unlikely to be appropriate to address all scenarios. However, harmonization of key considerations and assumptions can provide a consistent basis for developing the appropriate nonclinical safety development plans for individual programs, reducing uncertainty across regulatory regions and unnecessary animal use. Several key areas of nonclinical safety testing are nearing maturation for a harmonized approach, including species selection, certain aspects of study design, study duration, and unintended genomic integration risks. Furthermore, several emerging topics are unaddressed in current regulatory guidance for gene therapy products, which will become key areas of differentiation for the next generation of therapeutics. These topics include redosing, juvenile/pediatric safety, and reproductive/developmental safety testing, where relevant experience from other modalities can be applied. The rationale and potential study design considerations for these topics will be proposed, acknowledging that certain aspects of gene therapy development are not considered appropriate for harmonization. This article provides an overview of the current nonclinical safety regulatory landscape, summarizes typical nonclinical safety study designs, highlights areas of uncertainty, and discusses emerging topics that warrant consideration. Specific recommendations and perspectives are provided to inform future regulatory discussions and harmonization efforts.

Nonclinical development strategies for gene therapies are unique from other modalities. The European Federation of Pharmaceutical Industries and Associates (EFPIA) Gene Therapy Working Group surveyed EFPIA member and nonmember pharmaceutical and biotechnology companies about their current practices for designing and implementing nonclinical toxicology studies to support the development of viral vector-delivered in vivo gene therapies. Compiled responses from 17 companies indicated that these studies had some variability in species selection, study-design elements, biodistribution, immunogenicity or genomic insertion assessments, safety pharmacology, and regulatory interactions. Although there was some consistency in general practice, there were examples of extreme case-by-case differences. The responses and variability are discussed herein. Key development challenges were also identified. Results from this survey emphasize the importance for harmonization of regulatory guidelines for the development of gene-therapy products, while still allowing for case-by-case flexibility in nonclinical toxicology studies. However, the appropriate timing for a harmonized guidance, particularly with a platform that continues to rapidly evolve, remains in question., Graphical Abstract, The European Federation of Pharmaceutical Industries and Associates Gene Therapy Working Group conducted a survey on current practices associated with nonclinical toxicity studies conducted to support the development of in vivo viral vector-delivered gene therapies. Responses provide insight on studies conducted, endpoints evaluated, and challenges faced by biopharmaceutical/biotechnology companies.

Summary Aim To investigate the effect of ablative fractional CO2 laser (AFCO2L) on burns scar appearance and dermal architecture at 6 weeks and up to 3-years post-treatment. Methods Twenty adult patients with a burn-related scar were recruited. Inclusion criteria were a minimum scar area of 10 × 10 cm and Vancouver scar scale (VSS) score of >5 and ≥6 months since the time of injury. The region of scar was randomised to treatment/control zones. Treatment zones received 3 standardised laser treatments at 4- to 6-week intervals. All areas of scar received standard scar care. Outcome measures were recorded at baseline, 6-weeks post final treatment and up to 3 years post-treatment. Measures included blinded assessor VSS, Patient Scar Assessment Scale and histological tissue analysis. Results Nineteen and nine patients completed the short- and long-term studies, respectively. Clinical results revealed improvement in all scar areas over time. There was a statistically significant improvement in pain and itch in the treatment zone compared to the control zone at 6 weeks. Histological data revealed a significant increase in medium-sized collagen fibres at 6 weeks relative to the control site. Sub-group analysis according to scar age revealed greater histological improvement following laser treatment in immature scars relative to more mature scar. Conclusions Results demonstrate that 3 treatments of AFCO2L significantly improve scar pain, itch and dermal architecture at 6 weeks post-treatment. Histological results suggest greater potential in treating immature scar. Further investigation into the timing of laser treatment could help assist treatment protocols.

An important histological difference between normal, uninjured dermis and scar tissue such as that found in keloid scars is the pattern (morphological architecture) in which the collagen is deposited and arranged. In the uninjured dermis, collagen bundle architecture appears randomly organized (or in a basket weave formation), whereas in pathological conditions such as keloid scar tissue, collagen bundles are often found in whorls or in a hypotrophic scar collagen is more densely packed in a parallel configuration. In the case of skin, a scar disables the dermis, leaving it weaker, stiff and with a loss of optimal functionality. The absence of objective and quantifiable assessments of collagen orientation is a major bottleneck in monitoring progression of scar therapeutics. In this article, a novel quantitative approach for analyzing collagen orientation is reported. The methodology is demonstrated using collagen produced by cells in a model scar environment and examines collagen remodeling post-TGFβ stimulation in vitro. The method is shown to be reliable and effective in identifying significant coherency differences in the collagen deposited by human keloid scar cells. The technique is also compared for analysing collagen architecture in rat sections of normal, scarred skin and tendon tissue. Results demonstrate that the proposed computational method provides a fast and robust way of analyzing collagen orientation in a manner surpassing existing methods. This study establishes this methodology as a preliminary means of monitoring in vitro and in tissue treatment modalities which are expected to alter collagen morphology.

Insulin detemir has a different profile of action on the central nervous system (CNS) than human insulin. It has been hypothesized that this is caused by an altered ability of insulin detemir to cross the blood-brain barrier (BBB). Here, we measured the permeability of the BBB to insulin detemir. We labeled insulin detemir with radioactive iodine (I-Det) and examined its ability to cross the BBB of the mouse. Permeation was assessed after intravenous injection and by brain perfusion in the presence or absence of excess insulin detemir. The ability of insulin detemir to inhibit human insulin transport across the BBB was also assessed. I-Det did not cross the BBB either after intravenous injection or when studied by brain perfusion, a method which removes or reduces the influence of circulating proteins. Unlabeled detemir was about 10 times less potent than human insulin at inhibiting the transport of radioactive human insulin across the BBB. The altered CNS profile of insulin detemir may be caused by its poor access to CNS receptors and by a block of human insulin from crossing the BBB.

Purpose This study evaluated a new subretinal method for delivery of human or pig umbilical tissue-derived cells (hUTC or pUTC, respectively) using a novel subretinal injection cannula and suprachoroidal approach in Gottingen minipig eyes. hUTC (palucorcel) are currently under development for treating geographic atrophy in humans. Methods Twenty-four Gottingen minipigs (divided into eight groups) were subretinally administered palucorcel, pUTC, or vehicle. In some cases, fluorescently labeled cells and vehicle were administered. Conjunctival cutdown and sclerotomy were performed, then a flexible cannula containing a microneedle was inserted and advanced into the suprachoroidal space. The microneedle was deployed and visualized; 50 μL cells (target concentration, 11.2 × 106 cells/mL [560,000 cells/eye]) or vehicle was injected subretinally. Safety outcomes were evaluated. Results For all animals, cells and vehicle were successfully administered. Labeled cells or fluorescent vehicle were contained in the subretinal bleb, without leakage into the vitreous. No retinal detachment or vitreous traction band was identified by ophthalmologic examination. At all time points, observed microscopic changes were attributable to experimental procedures. On histopathology immediately after injection, localized retinal detachments were seen, along with focal retinal, choroidal, and/or scleral discontinuities. A moderate inflammatory response was seen in a limited number of animals. In the allogeneic setting, no antibody responses were detectable. Anti-human UTC antibodies were detected in the xenogeneic setting. Conclusions Palucorcel, pUTC, and vehicle were successfully administered to Gottingen minipigs using a novel subretinal injection cannula via a suprachoroidal surgical approach, with no significant adverse events; therefore, this technique appears to be feasible for further clinical development.

Objectives: The cytokines interleukin (IL)-1β and IL-6 are modulators of the neuroimmune axis and have been implicated in neuronal cell death cascades after ischemia or infection. Previous work has shown that some cross-species conservation exists between human and rodent blood-brain barrier (BBB) transport systems. To further assess cross-species conservation of cytokine transport across the BBB, the current studies investigated permeability and inhibition of ovine IL-1β and IL-6 in the mouse. Methods: IL-1β or IL-6 was radioactively labeled with 131I and injected into the jugular vein at time zero. A subset of mice received 1 or 3 µg/mouse of an unlabeled ovine or murine cytokine (IL-1β or IL-6) to assess self- and/or cross-inhibition of transport. Permeability was assessed using multiple-regression analysis. Results: There was a significant linear relationship for both ovine 131I-IL-1β and 131I-IL-6 between brain/serum ratios and exposure time, indicating BBB permeability. Inclusion of 3 µg/mouse unlabeled ovine IL-1β or IL-6 significantly reduced the transport of ovine 131I-IL-1β or 131I-IL-6, respectively, across the BBB. Transport of both ovine 131I-IL-1β and 131I-IL-6 was significantly inhibited by 1 µg/mouse of murine IL-1β or IL-6, respectively. In contrast, 1 µg/mouse of unlabeled ovine IL-1β or IL-6 did not inhibit the transport of murine 131I-IL-1β or 131I-IL-6. Conclusions: Ovine IL-1β and IL-6 cross the mouse BBB by saturable transport. Inhibition of transport by murine homologs indicates that both species use the same transport mechanisms. Conversely, an inability of ovine cytokines to significantly inhibit the transport of murine cytokines indicates that mouse BBB has a lower affinity for ovine than murine cytokines. Knowledge of species-conserved BBB transport mechanisms may facilitate the development of novel animal models of central nervous system pathogenesis.

Decreased clearance is the main reason amyloid-beta protein (Abeta) is increased in the brains of patients with Alzheimer's disease (AD). The neurovascular hypothesis states that this decreased clearance is caused by impairment of low density lipoprotein receptor related protein-1 (LRP-1), the major brain-to-blood transporter of Abeta at the blood-brain barrier (BBB). As deletion of the LRP-1 gene is a lethal mutation, we tested the neurovascular hypothesis by developing a cocktail of phosphorothioate antisenses directed against LRP-1 mRNA. We found these antisenses in comparison to random antisense selectively decreased LRP-1 expression, reduced BBB clearance of Abeta42, increased brain levels of Abeta42, and impaired learning ability and recognition memory in mice. These results support dysfunction of LRP-1 at the BBB as a mechanism by which brain levels of Abeta could increase and AD would be promoted.

Alzheimer's disease (AD) brains are characterized by accumulation of amyloid beta protein (Abeta) and neuroinflammation. Increased blood-to-brain influx and decreased brain-to-blood efflux across the blood-brain barrier (BBB) have been proposed as mechanisms for Abeta accumulation. Epidemiological studies suggest that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the progression of AD. We hypothesized that inflammation alters BBB handling of Abeta. Mice treated with lipopolysaccharide (LPS) had increased brain influx and decreased brain efflux of Abeta, recapitulating the findings in AD. Neither influx nor efflux was mediated by LPS acting directly on BBB cells. Increased influx was mediated by a blood-borne factor, indomethacin-independent, blocked by the triglyceride triolein, and not related to expression of the blood-to-brain transporter of Abeta, RAGE. Serum levels of IL-6, IL-10, IL-13, and MCP-1 mirrored changes in Abeta influx. Decreased efflux was blocked by indomethacin and accompanied by decreased protein expression of the brain-to-blood transporter of Abeta, LRP-1. LPS paradoxically increased expression of neuronal LRP-1, a major source of Abeta. Thus, inflammation potentially increases brain levels of Abeta by three mechanisms: increased influx, decreased efflux, and increased neuronal production.

Leptin, a pleiotropic adipokine, is a central regulator of appetite and weight and a key immunomodulatory protein. Although inborn leptin deficiency causes weight gain, it is unclear whether induced leptin deficiency in adult wild-type animals would be orexigenic. Previous work with a potent competitive leptin antagonist did not induce a true metabolic state of leptin deficiency in mice because of a short circulating half-life. In this study, we increased the half-life of the leptin antagonist by pegylation, which resulted in significantly increased bioavailability and retaining of antagonistic activity. Mice administered the pegylated antagonist showed a rapid and dramatic increase in food intake with weight gain. Resulting fat was confined to the mesenteric region with no accumulation in the liver. Serum cholesterol, triglyceride, and hepatic aminotransferases remained unaffected. Weight changes were reversible on cessation of leptin antagonist treatment. The mechanism of severe central leptin deficiency was found to be primarily caused by blockade of transport of circulating leptin across the blood-brain barrier with antagonisms at the arcuate nucleus playing a more minor role. Altogether we introduce a novel compound that induces central and peripheral leptin deficiency. This compound should be useful in exploring the involvement of leptin in metabolic and immune processes and could serve as a therapeutic for the treatment of cachexia.

Although pain was previously not considered an important element of multiple sclerosis (MS), recent evidence indicates that over 50% of MS patients suffer from chronic pain. In the present study, we utilized the Theiler's murine encephalomyelitis virus (TMEV) model of MS to examine whether changes in nociception occur during disease progression and to investigate whether sex influences the development of nociception or disease-associated neurological symptoms. Using the rotarod assay, TMEV infected male mice displayed increased neurological deficits when compared to TMEV infected female mice, which mimics what is observed in human MS. While both male and female TMEV infected mice exhibited thermal hyperalgesia and mechanical allodynia, female mice developed mechanical allodynia at a faster rate and displayed significantly more mechanical allodynia than male mice. Since neuropathic symptoms have been described in MS patients, we quantified sensory nerve fibers in the epidermis of TMEV-infected and non-infected mice to determine if there were alterations in epidermal nerve density. There was a significantly higher density of PGP9.5 and CGRP-immunoreactive axons in the epidermis of TMEV-infected mice versus controls. Collectively these results indicate that the TMEV model is well suited to study the mechanisms of MS-induced nociception and suggest that alterations in peripheral nerve innervation may contribute to MS pain.

Multiple sclerosis patients typically experience increased pain that is relatively insensitive to opiate treatment. The mechanistic basis for this increased nociception is currently poorly understood. In the present study, we utilized the Theiler's murine encephalomyelitis virus (TMEV) model of MS to examine possible changes in spinal cord opioid receptor mRNA over the course of disease progression. TMEV infection led to significantly decreased mu, delta and kappa opioid receptor mRNA expression as analyzed by quantitative real-time PCR in both male and female mice at days 90, 150 and 180 post-infection (PI). Since opioid receptor mRNA expression decreased in TMEV mice, we examined whether opiate analgesia is also altered. TMEV infected female mice had significantly decreased opiate analgesia in thermal nociceptive tests beginning at day 90 PI, while TMEV-infected male mice did not display significantly decreased opiate analgesia until day 120 PI. The novel finding that opioid receptor expression is significantly decreased in the spinal cord of TMEV mice could explain the increased nociception and loss of opiate analgesia observed in both TMEV mice and multiple sclerosis patients.

Microarrays are a powerful tool for comparison and understanding of gene expression levels in healthy and diseased states. The method relies upon the assumption that signals from microarray features are a reflection of relative gene expression levels of the cell types under investigation. It has previously been reported that the classical fluorescent dyes used for microarray technology, Cy3 and Cy5, are not ideal due to the decreased stability and fluorescence intensity of the Cy5 dye relative to the Cy3, such that dye bias is an accepted phenomena necessitating dye swap experimental protocols and analysis of differential dye affects. The incentive to find new fluorophores is based on alleviating the problem of dye bias through synonymous performance between counterpart dyes. Alexa Fluor 555 and Alexa Fluor 647 are increasingly promoted as replacements for CyDye in microarray experiments. Performance relates to the molecular and steric similarities, which will vary for each new pair of dyes as well as the spectral integrity for the specific application required. Comparative analysis of the performance of these two competitive dye pairs in practical microarray applications is warranted towards this end. The findings of our study showed that both dye pairs were comparable but that conventional CyDye resulted in significantly higher signal intensities (P0.05) and signal minus background levels (P0.05) with no significant difference in background values (P0.05). This translated to greater levels of differential gene expression with CyDye than with the Alexa Fluor counterparts. However, CyDye fluorophores and in particular Cy5, were found to be less photostable over time and following repeated scans in microarray experiments. These results suggest that precautions against potential dye affects will continue to be necessary and that no one dye pair negates this need.

Amyloid beta protein (Abeta) in Alzheimer's disease induces oxidative stress through several mechanisms, including stimulation of nitric oxide synthase (NOS) activity. We examined NOS activity and expression in the senescence-accelerated mouse P8 (SAMP8) line. The SAMP8 strain develops with aging cognitive impairments, increases in Abeta, and oxidative stress, all reversed by amyloid precursor protein antisense or Abeta antibody treatment. We found here that hippocampal NOS activity in 12-month-old SAMP8 mice was nearly double that of 2-month-old SAMP8 or CD-1 mice, but with no change in NOS isoenzyme mRNA and protein levels. Antisense or antibody treatment further increased NOS activity in aged SAMP8 mice. Antisense treatment increased inducible NOS (iNOS) mRNA levels, decreased neuronal NOS mRNA and protein levels, but did not affect endothelial NOS (eNOS) or iNOS protein or eNOS mRNA levels. These results suggest a complex relation between Abeta and NOS in the SAMP8 that is largely mediated through posttranslational mechanisms.

The blood-brain barrier (BBB) mediates interactions between the immune and central nervous systems in several ways and is central to many mechanisms that form communication pathways within the neuroimmune axis. Here, we briefly review the chief types of interactions. Cytokines and immune cells cross the BBB by regulated mechanisms. Cytokines alter BBB characteristics, including the integrity of the BBB, its transport systems, and its ability to control immune cell trafficking. The cells that comprise the BBB secrete cytokines, prostaglandins, nitric oxide, and other immuneactive factors. Such secretion is both constituitive and inducible, depending on the substance secreted. Secretion is also polarized; that is, secretion can be from either the luminal or abluminal membrane. This raises the possibility that the BBB may recieve signal at one membrane and secrete cytokine from the other as a mechanism of communication within the neuroimmune axis. In brief, the BBB is a central player in a number of mechanisms and pathways that comprise the neuroimmune axis.

P-glycoprotein (P-gp) is a brain-to-blood efflux system that controls the ability of many drugs and endogenous substances to access the brain. In vitro work has shown that inflammatory states mediated through lipopolysaccharide (LPS) and tumor necrosis factor-alpha first impair and then stimulate P-gp activity. Here, we determined whether LPS can affect P-gp function in vivo. Mice treated with a single intraperitoneal injection of LPS (3 mg/kg) showed an inhibition of P-gp function. As assessed by brain perfusion, inhibition began 18 h after LPS administration and lasted until 36 h after administration. P-gp protein was increased by 44%, consistent with P-gp inhibition occurring through post-translational mechanisms. Unlike other effects of LPS on blood–brain barrier function, neither nitric oxide nor prostaglandin inhibition had an effect. We conclude that induction of proinflammatory states as exemplified by LPS treatment can inhibit P-gp function in vivo at the blood–brain barrier.

Insulin transported across the blood-brain barrier (BBB) has many effects within the central nervous system. Insulin transport is not static but altered by obesity and inflammation. Lipopolysaccharide (LPS), derived from the cell walls of Gram-negative bacteria, enhances insulin transport across the BBB but also releases nitric oxide (NO), which opposes LPS-enhanced insulin transport. Here we determined the role of NO synthase (NOS) in mediating the effects of LPS on insulin BBB transport. The activity of all three NOS isoenzymes was stimulated in vivo by LPS. Endothelial NOS and inducible NOS together mediated the LPS-enhanced transport of insulin, whereas neuronal NOS (nNOS) opposed LPS-enhanced insulin transport. This dual pattern of NOS action was found in most brain regions with the exception of the striatum, which did not respond to LPS, and the parietal cortex, hippocampus, and pons medulla, which did not respond to nNOS inhibition. In vitro studies of a brain endothelial cell (BEC) monolayer BBB model showed that LPS did not directly affect insulin transport, whereas NO inhibited insulin transport. This suggests that the stimulatory effect of LPS and NOS on insulin transport is mediated through cells of the neurovascular unit other than BECs. Protein and mRNA levels of the isoenzymes indicated that the effects of LPS are mainly posttranslational. In conclusion, LPS affects insulin transport across the BBB by modulating NOS isoenzyme activity. NO released by endothelial NOS and inducible NOS acts indirectly to stimulate insulin transport, whereas NO released by nNOS acts directly on BECs to inhibit insulin transport.

Interleukin-1alpha (IL-1alpha), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-alpha) are proinflammatory cytokines with potent neuromodulatory effects and are implicated in the etiology and pathogenesis of various psychological and neurological disorders. The findings that chronic morphine treatment alters both blood-brain barrier (BBB) function and cytokine production raises the possibility that morphine can also modulate cytokine transport across the BBB. Here, we found that acute morphine treatment (12 mg/kg i.p.) did not alter blood-to-brain transport of IL-1alpha, IL-2 or TNF-alpha. Whereas chronic morphine treatment (48 h after implantation of 75 mg morphine pellets) and withdrawal from morphine (10-15 min after an i.p. injection of 1mg/kg of naltroxone 48 h after implantation of 75 mg morphine pellets) did not alter blood-to-brain transport of IL-1alpha or TNF-alpha, both the chronic morphine treatment and withdrawal from morphine groups had increased blood-to-brain transport of IL-2. Typically, the permeability of the BBB to IL-2 is dominated by brain-to-blood efflux, with only limited blood-to-brain transport. Here, we found that chronic morphine and withdrawal from morphine did not alter brain-to-blood efflux, but induced a novel saturable blood-to-brain transport system. Whereas IL-1alpha, IL-2, and TNF-alpha are all proinflammatory cytokines, morphine exposure has individualized effects on their blood-to-brain transport.

Microarray technology is readily available to scientists interested in gene expression. Commensurate with this availability is the growing market in accessory products offering convenience but potentially variable performance. Here we evaluate seven commercial kits for probe labeling against a human apoptosis oligonucleotide array. All kits were found to label probes successfully using the manufacturers’ instructions. The Stratagene Fairplay Microarray Labeling Kit was the most sensitive, with an overall call rate of 74% and the lowest rate of indeterminant calls for the HEK and HepG2 cell lines. The Invitrogen SuperScript Indirect cDNA Labeling System showed the most reproducible gene expression pattern and the least technical variation, both in terms of signal strength and between replicates on each array. The Promega Pronto! Plus System showed the least dye bias however, a higher level of variation between replicates was observed. Pairwise comparisons revealed that the Promega Pronto! Plus System and Invitrogen SuperScript Indirect cDNA Labeling System had the most similarity in their patterns of gene expression. Results obtained suggest variability in the performance of commercial kits between different manufacturers. This study supports the need to conduct comparative evaluations of commercial microarray probe labeling kits and the need for validation prior to use.

Corrigendum to ‘‘Lipopolysaccharide alters the blood–brain barrier transport of amyloid b protein: A mechanism for inflammation in the progression of Alzheimer’s disease’’ [Brain Behav. Immun. 23 (2009) 507–517] Laura B. Jaeger , Shinya Dohgu , Rukhsana Sultana , Jessica L. Lynch , Joshua B. Owen , Michelle A. Erickson , Gul N. Shah , Tulin O. Price , Melissa A. Fleegal-Demotta , D. Allan Butterfield , William A. Banks a,b,c,⇑

Microarray is a widely used technique to study gene expression. The increasing interest in the technology has resulted in increased availability of commercial accessory reagents and instrumentation. In principle, commercial advances in reagents, kits and equipment should greatly improve assay performance, since they each bring a measure of quality assurance and uniformity to the data above that which may be obtained from the original manual hybridization processes. However, independent validation of this perceived benefit remains an essential part of the adoption process and, in microarrays, this has often been overlooked for want of immediate convenience. We describe here the comparative evaluation of two automated hybridization instruments, namely the MAUI(R) (microarray user interface) hybridization system and the GeneTAC HybStation, against the conventional manual coverslip hybridization methodology. Our results show that there is a significant advantage in using automated hybridization instrumentation over the diffusion-based coverslip methodology. We observed an enhancement of the mean signal, signal-to-noise ratio, and reproducibility between replicates when using both the MAUI(R) hybridization system and the GeneTAC HybStation. Automation further reduced labour time, offered simplicity and greater reproducibility and accuracy in the results. The present study has independently validated the benefits automation brings to the microarray hybridization process and highlights the differences between the instruments examined. We further comment on the higher quality of spot morphology when hybridization is performed at a lower temperature in combination with our buffer of choice.